High yield transient expression in mammalian cells using unique pairing of high density growth and transfection medium and expression enhancers

ABSTRACT

High-Yield mammalian transient expression systems can include a cell culture media (particularly serum free, non animal derived, and/or chemically defined media) for introducing macromolecules and compounds (e.g., nucleic acid molecules) into cells (e.g., eukaryotic cells). Cells containing such introduced materials can then be cultured in the cell culture media. In particular, the invention allows introduction of nucleic acid molecules (e.g., vectors) into cells (particularly mammalian cells) and expression of proteins encoded by the nucleic acid molecules in the cells. The invention obviates the need to change the cell culture medium each time a different procedure is performed with the cells (e.g., culturing cells vs. transfecting cells). The invention also relates to compositions and kits useful for culturing and transforming/transfecting cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the right of priority under 35 U.S.C. § 119(e)to U.S. Provisional Application Ser. No. 61/641,864, filed May 2, 2012,which is commonly owned with the present application and which theentire contents thereof are hereby expressly incorporated by referenceas though fully set forth herein.

FIELD OF THE INVENTION

The present invention generally relates to the fields of transfectionand cell culture. In particular, the present invention provides atransfection system suitable for yield expression of recombinantproteins in cultured mammalian cells. The invention further related tosystems and methods for high yield expression of recombinant proteins inmammalian cells.

BACKGROUND

Cell culture media provide the nutrients necessary to maintain and growcells in a controlled, artificial and in vitro environment.Characteristics and formulations of cell culture media vary dependingupon the particular cellular requirements. Important parameters includeosmolarity, pH, and nutrient compositions.

Cell culture medium formulations have been well documented in theliterature and a large number of media are commercially available. Inearly cell culture work, medium formulations were based upon thechemical composition and physicochemical properties (e.g., osmolality,pH, etc.) of blood and were referred to as “physiological solutions”(Ringer, S., J. Physiol. 3:380-393 (1880); Waymouth, C., In: Cells andTissues in Culture, Vol. 1, Academic Press, London, pp. 99-142 (1965);Waymouth, C., In Vitro 6:109-127 (1970)). However, cells in differenttissues of a mammalian body are exposed to different microenvironmentswith respect to oxygen/carbon dioxide partial pressure andconcentrations of nutrients, vitamins, and trace elements; accordingly,successful in vitro culture of different cell types may require the useof different medium formulations. Typical components of cell culturemedia include amino acids, organic and inorganic salts, vitamins, tracemetals, sugars, lipids and nucleic acids, the types and amounts of whichmay vary depending upon the particular requirements of a given cell ortissue type.

Medium formulations have been used to cultivate a number of cell typesincluding animal, plant and bacterial cells. Cultivated cells have manyuses including the study of physiological processes and the productionof useful biological substances. Examples of such useful productsinclude monoclonal antibodies, hormones, growth factors, enzymes and thelike. Such products have many commercial and therapeutic applicationsand, with the advent of recombinant DNA technology, cells can beengineered to produce large quantities of these products. Cultured cellsare also routinely used for the isolation, identification and growth ofviruses that can be used as vectors and/or vaccines. Thus, the abilityto cultivate cells in vitro is not only important for the study of cellphysiology, but is also necessary for the production of usefulsubstances that may not otherwise be obtained by cost-effective means.

Among the various cell types that have been grown using in vitro cellculture media, of particular interest are cells derived from theepithelium. The epithelium lines the internal and external surfaces ofthe organs and glands of higher organisms. Because of this localizationat the external interface between the environment and the organism(e.g., the skin) or at the internal interface between an organ and theinterstitial space (e.g., the intestinal mucosal lining), the epitheliumhas a major role in the maintenance of homeostasis. The epitheliumcarries out this function, for example, by regulating transport andpermeability of nutrients and wastes (Freshney, R. I., in: Culture ofEpithelial Cells, Freshney, R. I., ed., New York: Wiley-Liss, pp. 1-23(1992)).

The cells making up the epithelium are generically termed epithelialcells. These cells can be present in multiple layers as in the skin, orin a single layer as in the lung alveoli. As might be expected, thestructure, function and physiology of epithelial cells are oftentissue-specific. For example, the epidermal epithelial cells of the skinare organized as stratified squamous epithelium and are primarilyinvolved in forming a protective barrier for the organism, while thesecretory epithelial cells of many glands are often found in singlelayers of cuboidal cells that have a major role in producing secretoryproteins and glycoproteins. Regardless of their location or function,however, epithelial cells are usually regenerative. That is, undernormal conditions, or in response to injury or other activatingstimulus, epithelial cells are capable of dividing or growing. Thisregenerative capacity has facilitated the in vitro manipulation ofepithelial cells, to the point where a variety of primary epithelialcells and cell lines have been successfully cultivated in vitro(Freshney, Id.).

While the isolation and use of a variety of epithelial cells andepithelial cell lines have been reported in the literature, the humanembryonic kidney cell line 293 (“293 cells”), which exhibits epithelialmorphology, has proven particularly useful for studies of the expressionof exogenous ligand receptors, production of viruses and expression ofallogeneic and xenogeneic recombinant proteins. For example, U.S. Pat.No. 5,166,066 describes the construction of a stable 293 cell linecomprising functional GABA receptors that include a benzodiazepinebinding site that have proven useful in identification and screening ofcandidate psychoactive drugs. 293 cells have also been used to produceviruses such as natural and recombinant adenoviruses (Gamier, A., etal., Cytotechnol. 15:145-155 (1994); Bout, A., et al., Cancer GeneTherapy 3(6):S24, abs. P-52 (1996); Wang, J.-W., et al., Cancer GeneTherapy 3(6):S24, abs. P-53 (1996)), which can be used for vaccineproduction or construction of adenovirus vectors for recombinant proteinexpression. Finally, 293 cells have proven useful in large-scaleproduction of a variety of recombinant human proteins (Berg, D. T., etal., BioTechniques 14(6):972-978 (1993); Peshwa, M. V., et al.,Biotechnol. Bioeng. 41:179-187 (1993); Gamier, A., et al., Cytotechnol.15:145-155 (1994)).

Cells loosely called fibroblasts have been isolated from many differenttissues and are understood to be connective tissue cells. It is clearlypossible to cultivate cell lines, loosely termed fibroblastic cells,from embryonic and adult tissues. Fibroblasts characteristically have a“spindle” appearance. Fibroblast-like cells have morphologicalcharacteristics typical of fibroblast cells. Under a light microscopethe cells appear pointed and elongated (“spindle shaped”) when they growas a monolayer on the surface of a culture vessel. Cell lines can beregarded as fibroblast or fibroblast-like after confirmation withappropriate markers, such as collagen, type I ((Freshney, R. I., in:Culture of Epithelial Cells, Freshney, R. I., ed., New York: Wiley-Liss,pp. 1-23 (1987)).

CHO cells have been classified as both epithelial and fibroblast cellsderived from the Chinese hamster ovary. A cell line started from Chinesehamster ovary (CHO-K1) (Kao, F.-T. And Puck, T. T., Proc. Natl. Acad.Sci. USA 60:1275-1281 (1968) has been in culture for many years but itsidentity is still not confirmed.

Most primary mammalian epithelial cells, mammalian fibroblast cells,epithelial cell lines, and fibroblast cell lines are typically grown inmonolayer culture. For some applications, however, it would beadvantageous to cultivate such cells as suspension cultures. Forexample, suspension cultures grow in a three-dimensional space.Monolayer cultures in similar-sized vessels, however, can only growtwo-dimensionally on the vessel surface. Thus, suspension cultures canresult in higher cell yields and, correspondingly, higher yields ofbiologicals (e.g., viruses, recombinant polypeptides, etc.) compared tomonolayer cultures. In addition, suspension cultures are often easier tofeed and scale-up, via simple addition of fresh culture media (dilutionsubculturing) to the culture vessel rather than trypsinization andcentrifugation as is often required with monolayer cultures. The ease offeeding and the ease with which suspension cultures can be scaled uprepresent a substantial saving in time and labor for handling acomparable number of cells.

Many anchorage-dependent cells, such as primary epithelial cells,primary fibroblast cells, epithelial cell lines, and fibroblast celllines, however, are not easily adapted to suspension culture. Since theyare typically dependent upon anchorage to a substrate for optimalgrowth, growth of these cells in suspension can require their attachmentto microcarriers such as latex or collagen beads. Thus, cells grown inthis fashion, while capable of higher density culture than traditionalmonolayer cultures, are still technically attached to a surface;subculturing of these cells therefore requires similar steps as thoseused for the subculturing of monolayer cultures. Furthermore, when largebatch or fermenter cultures are established, a large volume ofmicrocarriers often settles to the bottom of the culture vessel, therebyrequiring a more complicated agitation mechanism to keep themicrocarriers (and thus, the cells) in suspension without causing sheardamage to the cells (Peshwa, M. V., et al., Biotechnol. Bioeng.41:179-187 (1993)).

Although many transformed cells are capable of being grown in suspension(Freshney, R. I., Culture of Animal Cells: A Manual of Basic Technique,New York: Alan R. Liss, Inc., pp. 123-125 (1983)), successful suspensioncultures often require relatively high-protein media or supplementationof the media with serum or serum components (such as the attachmentfactors fibronectin and/or vitronectin), or sophisticated perfusionculture control systems (Kyung, Y.-S., et al., Cytotechnol. 14:183-190(1994)), which can be disadvantageous. In addition, many epithelialcells when grown in suspension form aggregates or “clumps” which caninterfere with successful subculturing and reduce growth rate andproduction of biologicals by the cultures. When clumping occurs, theoverall cellular surface area exposed to medium is decreased and thecells are deprived of nutrition and are unable to efficiently exchangewaste into the medium. As a result, growth slows, diminished celldensities are obtained, and protein expression is compromised.

Typically, cell culture media formulations are supplemented with a rangeof additives, including undefined components such as fetal bovine serum(FBS) (5-20% v/v) or extracts from animal embryos, organs or glands(0.5-10% v/v). While FBS is the most commonly applied supplement inanimal cell culture media, other serum sources are also routinely used,including newborn calf, horse and human. Organs or glands that have beenused to prepare extracts for the supplementation of culture mediainclude submaxillary gland (Cohen, S., J. Biol. Chem. 237:1555-1565(1961)), pituitary (Peehl, D. M., and Ham, R. G., In Vitro 16:516-525(1980); U.S. Pat. No. 4,673,649), hypothalamus (Maciag, T., et al.,Proc. Natl. Acad. Sci. USA 76:5674-5678 (1979); Gilchrest, B. A., etal., J. Cell Physiol. 120:377-383 (1984)), ocular retina (Barretault,D., et al., Differentiation 18:29-42 (1981)) and brain (Maciag, T., etal., Science 211:1452-1454 (1981)). These types of chemically undefinedsupplements serve several useful functions in cell culture media(Lambert, K. J. et al., In: Animal Cell Biotechnology, Vol. 1, Spier, R.E. et al., Eds., Academic Press New York, pp. 85-122 (1985)). Forexample, these supplements provide carriers or chelators for labile orwater-insoluble nutrients; bind and neutralize toxic moieties; providehormones and growth factors, protease inhibitors and essential, oftenunidentified or undefined low molecular weight nutrients; and protectcells from physical stress and damage. Thus, serum or organ/glandextracts are commonly used as relatively low-cost supplements to providean optimal culture medium for the cultivation of animal cells.

Unfortunately, the use of serum or organ/gland extracts in tissueculture applications has several drawbacks (Lambert, K. J. et al., In:Animal Cell Biotechnology, Vol. 1, Spier, R. E. et al., Eds., AcademicPress New York, pp. 85-122 (1985)). For example, the chemicalcompositions of these supplements and sera vary between lots, even froma single manufacturer. The supplements can also be contaminated withinfectious agents (e.g., mycoplasma and viruses) which can seriouslyundermine the health of the cultured cells and the quality of the finalproduct. The use of undefined components such as serum or animalextracts also prevents the true definition and elucidation of thenutritional and hormonal requirements of the cultured cells, thuseliminating the ability to study, in a controlled way, the effect ofspecific growth factors or nutrients on cell growth and differentiationin culture. Moreover, undefined supplements prevent the researcher fromstudying aberrant growth and differentiation and the disease-relatedchanges in cultured cells. Finally and most importantly to thoseemploying cell culture media in the industrial production of biologicalsubstances, serum and organ/gland extract supplementation of culturemedia can complicate and increase the costs of the purification of thedesired substances from the culture media due to nonspecificco-purification of serum or extract proteins.

Improved levels of recombinant protein expression are obtained fromcells grown in serum-free medium, relative to the level of expressionseen in cells grown in medium supplemented with serum (Battista, P. J.et al., Am. Biotech. Lab. 12:64-68 (1994)). However, serum-free mediacan still contain one or more of a variety of animal-derived components,including albumin, fetuin, various hormones and other proteins. Thepresence of proteins or peptides makes purification of recombinantprotein difficult, time-consuming, and expensive.

To overcome these drawbacks of the use of serum or organ/gland extracts,a number of so-called “defined” media have been developed. These media,which often are specifically formulated to support the culture of asingle cell type, contain no undefined supplements and insteadincorporate defined quantities of purified growth factors, proteins,lipoproteins and other substances usually provided by the serum orextract supplement. Since the components (and concentrations thereof) insuch culture media are precisely known, these media are generallyreferred to as “defined culture media.” Sometimes used interchangeablywith “defined culture media” is the term “serum-free media” or “SFM.” Anumber of SFM formulations are commercially available, such as thosedesigned to support the culture of endothelial cells, keratinocytes,monocytes/macrophages, lymphocytes, hematopoietic stem cells,fibroblasts, chondrocytes or hepatocytes which are available from LifeTechnologies Corporation, Carlsbad, Calif. The distinction between SFMand defined media, however, is that SFM are media devoid of serum andprotein fractions (e.g., serum albumin), but not necessarily of otherundefined components such as organ/gland extracts. Indeed, several SFMthat have been reported or that are available commercially contain suchundefined components, including several formulations supporting in vitroculture of keratinocytes (Boyce, S. T., and Ham, R. G., J. Invest.Dermatol. 81:33 (1983); Wille, J. J., et al., J. Cell. Physiol. 121:31(1984); Pittelkow, M. R., and Scott, R. E., Mayo Clin. Proc. 61:771(1986); Pirisi, L., et al., J. Virol. 61:1061 (1987); Shipley, G. D.,and Pittelkow, M. R., Arch. DermatoL 123:1541 (1987); Shipley, G. D., etal., J. Cell. Physiol. 138:511-518 (1989); Daley, J. P., et al., FOCUS(GIBCO/LTI) 12:68 (1990); U.S. Pat. Nos. 4,673,649 and 4,940,666). SFMthus cannot be considered to be defined media in the true definition ofthe term.

Defined media generally provide several distinct advantages to the user.For example, the use of defined media facilitates the investigation ofthe effects of a specific growth factor or other medium component oncellular physiology, which can be masked when the cells are cultivatedin serum- or extract-containing media. In addition, defined mediatypically contain much lower quantities of protein (indeed, definedmedia are often termed “low protein media”) than those containing serumor extracts, rendering purification of biological substances produced bycells cultured in defined media far simpler and less expensive.

Some extremely simple defined media, which consist essentially ofvitamins, amino acids, organic and inorganic salts and buffers have beenused for cell culture. Such media (often called “basal media”), however,are usually seriously deficient in the nutritional content required bymost animal cells. Accordingly, most defined media incorporate into thebasal media additional components to make the media more nutritionallycomplex, but to maintain the serum-free and low protein content of themedia. Examples of such components include bovine serum albumin (BSA) orhuman serum albumin (HSA); certain growth factors derived from natural(animal) or recombinant sources such as epidermal growth factor (EGF) orfibroblast growth factor (FGF); lipids such as fatty acids, sterols andphospholipids; lipid derivatives and complexes such asphosphoethanolamine, ethanolamine and lipoproteins; protein and steroidhormones such as insulin, hydrocortisone and progesterone; nucleotideprecursors; and certain trace elements (reviewed by Waymouth, C., in:Cell Culture Methods for Molecular and Cell Biology, Vol. 1: Methods forPreparation of Media, Supplements, and Substrata for Serum-Free AnimalCell Culture, Barnes, D. W., et al., eds., New York: Alan R. Liss, Inc.,pp. 23-68 (1984), and by Gospodarowicz, D., Id., at pp 69-86 (1984)).

The use of animal protein supplements in cell culture media, however,also has certain drawbacks. For example, there is a risk that theculture medium and/or products purified from it can be immunogenic,particularly if the supplements are derived from an animal differentfrom the source of the cells to be cultured. If biological substances tobe used as therapeutics are purified from such culture media, certainamounts of these immunogenic proteins or peptides can be co-purified andcan induce an immunological reaction, up to and including anaphylaxis,in an animal receiving such therapeutics.

To obviate this potential problem, supplements derived from the samespecies as the cells to be cultured can be used. For example, culture ofhuman cells can be facilitated using HSA as a supplement, while mediafor the culture of bovine cells would instead use BSA. This approach,however, runs the risks of introducing contaminants and adventitiouspathogens into the culture medium (such as Creutzfeld-Jakob Disease(CJD) from HSA preparations, or Bovine Spongiform Encephalopathy (“MadCow Disease”) prion from BSA preparations), which can obviouslynegatively impact the use of such media in the preparation of animal andhuman therapeutics. In fact, for such safety reasons, the biotechnologyindustry and government agencies are increasingly regulating,discouraging and even forbidding the use of cell culture mediacontaining animal-derived proteins which can contain such pathogens.

To overcome the limitations of the use of animal proteins in SFM,several attempts have been made to construct animal cell culture mediathat are completely free of animal proteins. For example, some culturemedia have incorporated extracts of yeast cells into the basal medium(see, for example, U.K. Patent Application No. GB 901673; Keay, L.,Biotechnol. Bioengin. 17:745-764 (1975)) to provide sources of nitrogenand other essential nutrients. In another approach, hydrolysates ofwheat gluten have been used, with or without addition of yeast extract,to promote in vitro growth of animal cells (Japanese Patent ApplicationNo. JP 2-49579). Still other media have been developed in which serum isreplaced by enzymatic digests of meat, or of proteins such asα-lactalbumin or casein (e.g., peptone), which have been traditionallyused in bacterial culture (Lasfargues, E. Y., et al., In Vitro8(6):494-500 (1973); Keay, L., Biotechnol. Bioeng. 17:745-764 (1975);Keay, L., Biotechnol. Bioeng. 19:399-411 (1977); Schlager, E.-J., J.Immunol. Meth. 194:191-199 (1996)). None of these approaches, however,provided a culture medium optimal for the cultivation of a variety ofanimal cells. Moreover, extracts from certain plants, including wheat,barley, rye and oats have been shown to inhibit protein synthesis incell-free systems derived from animal cells (Coleman, W. H., andRoberts, W. K., Biochim. Biophys. Acta 696:239-244 (1982)), suggestingthat the use of peptides derived from these plants in cell culture mediacan actually inhibit, rather than stimulate, the growth of animal cellsin vitro. More recently, animal cell culture SFM formulations comprisingrice peptides have been described and shown to be useful in cultivationof a variety of normal and transformed animal cells (see U.S. Pat. No.6,103,529, incorporated herein by reference in its entirety).

Notwithstanding the potential difficulties posed by the addition ofanimal derived supplements to cell culture media, such supplements arein routine use. One such supplement that is frequently added to definedmedia is transferrin. Transferrin functions in vivo to deliver iron tocells. The mechanism of iron uptake by mammalian cells has been reviewed(Qian, Z. M. and Tang, P. L. (1995) Biochim. Biophys. Acta 1269,205-214). As iron is required as a co-factor in numerous metabolicprocesses including energy generation and oxidative respiration,serum-free media are often supplemented with transferrin in order todeliver the requisite iron for the successful cultivation of most cellsin vitro. Concern about various potential adventitious agents inpreparations of transferrin has stimulated a search for other naturaliron carrier compounds which can be used as a substitute fortransferrin. This search is complicated by the fact that the naturaliron carriers are often derived from serum and thus are subject to theabove-described limitations of serum supplementation.

To overcome the limitations of using naturally derived metal carriers,certain metal binding compounds are being explored for use in supplyingmetals, particularly zinc, iron, manganese and magnesium, to culturedcells. Simple carriers such as chelating agents (e.g., EDTA) and certainacids or salts thereof (e.g., citrate, picolinate, and derivatives ofbenzoic acid or hydroxamic acid) have been shown to be useful in certainserum-free growth media (see U.S. Pat. Nos. 5,045,454 and 5,118,513;Testa et al., Brit. J. Haematol. 60:491-502, (1985); Ganeshaguru et al.,Biochem. Pharmacol. 29:1275-1279 (1980); White et al., Blood 48:923-929(1976)).

Although these references disclose some metal carriers, theinterpretation of the data is complicated by several experimentalfactors. The data were gathered from a limited number of cell lines andshow results of a single passage. In addition, the media weresupplemented with serum. Serum inherently contains transferrin and otherpotential iron carriers. There is a “carry-over effect” on growth ofcells which have been cultured in serum-supplemented medium, even afterone or two passages in the absence of serum or transferrin (see, forexample, Keenan, J. and Clynes, M. (1996) In Vitro Cell Dev. Biol-Animal32, 451-453). Other known metal binding compounds have been usedmedicinally to remove iron from the body and not for delivery.Unfortunately, many of these simple iron chelating compounds do notprovide sufficient iron availability to, or uptake by, cultured cells.

Once a suitable medium formulation for the growth of a particular celltype has been determined, it is frequently necessary to alter the cellin question so as to optimize the production of a desired biologicalsubstance. A critical step in the effective production and purificationof biological substances is the introduction of one or moremacromolecules (e.g., peptides, proteins, nucleic acids, etc.) into thecell in which the material will be produced. This can be accomplished bya variety of methods. One widely used method to introduce macromoleculesinto a cell is known as transfection.

Typically, the target cell is grown to a desired cell density in a cellculture medium optimized for growth of the cell. Once the desireddensity is reached, the medium is exchanged for a medium optimized forthe transfection process. Under most circumstances, the medium used fortransfection does not support the growth of the cells but thetransfection medium is merely used for the purpose of introducingnucleic acids into the cells. As a result, the process generallyrequires collecting the cells from the culture, usually bycentrifugation, washing the cells to remove traces of the growth medium,suspending the cells in a transfection medium in the presence of themacromolecule of interest, incubating the cells in the transfectionmedium for a period of time sufficient for the uptake of themacromolecule, optionally, removing the transfection medium and washingthe remnants of the transfection medium from the cells and thenre-suspending the transfected cells in a growth medium. The steps ofexchanging the growth media for transfection media, washing the cells,and exchanging the transfection media back to a growth media require agreat deal of hands-on manipulation of the cells thereby addingsubstantially to the time and expense of recombinant DNA technology.

As an historical example, 293 cells have been cultivated in monolayercultures in a serum-supplemented version of a complex medium (i.e.,DMEM). When grown in suspension, 293 cells have a tendency to aggregateinto large clusters of cells. The formation of these large cellaggregates reduces the viability of the cells. Since the cells in thecenter of the aggregates are not directly exposed to the medium, thesecells have limited access to nutrients in the medium and have difficultyin exchanging waste into the medium. In addition, this reduced access tothe medium makes cells in clusters unsuitable for genetic manipulationby factors introduced into the medium (i.e., for transformation bynucleic acids). As a result of these difficulties, 293 cells have notgenerally been used in suspension culture for the production ofbiological materials.

Thus, there still remains a need in the art for a cell medium andtransient transfection system that permits the growth of eukaryoticcells in suspension while permitting the transfection of the cells witha reduced amount of manipulation. Such a medium should preferably be aserum-free and/or chemically defined and/or protein-free medium and/or amedium lacking animal derived materials which facilitates the growth ofmammalian cells to high density and/or increases the level of expressionof recombinant protein, reduces cell clumping, and which does notrequire supplementation with animal proteins, such as serum,transferrin, insulin and the like. Preferably a medium of this type willpermit the suspension cultivation of mammalian cells that are normallyanchorage-dependent, including epithelial cells and fibroblast cells,such as 293 cells and CHO cells. Preferably, such a medium would alsoenable cultivation and culturing of the aforementioned cell types athigher density than can be typically obtained with currently availablemedia. Additionally, such culture media will allow easier and morecost-effective and efficient production and purification of highquantities of commercially or scientifically important biologicalsubstances (e.g., viruses, recombinant proteins, biologics, recombinantantibodies, etc.) produced by cultured mammalian cells in thebiotechnology industry, and will provide more consistent results inmethods employing the cultivation of mammalian cells. These and otherneeds are met by the present invention.

SUMMARY

The present invention provides a cell culture and transfection system,whereby the system supports introduction by way of transfection andsubsequent expression of one or more macromolecules (such as, e.g.,expressible nucleic acids) into a plurality of eukaryotic cells inculture, and further supports the cultivation and growth of the cellssubsequent to the introduction/transfection, wherein growth of the atleast one cell continues in the medium in the absence of the mediumbeing supplemented with fresh medium.

In some embodiments, it is not necessary to remove, replenish or replacethe medium used during the introduction/transfection of the cells fromthe presence of the cells to support the further growth thereof. Inanother preferred embodiment, after the introduction/transfection,growth of the cells and production of an expressed protein from theexpressible nucleic acid can be accomplished in a volume of medium thatis about the same volume up to no more than about 10 times the volume ofthe medium in which the introduction/transfection occurred. Using themedium of the present invention, it is not necessary to replenish,replace or supplement the medium after one has introduced nucleic acidinto cells, and before cells into which nucleic acid has been introducedare further cultured to express the nucleic acid.

Transient expression is fast becoming the system of choice for rapidmammalian protein production. The flexibility of transient transfectionenables a rapid realization time from concept to protein-in-hand andmany different proteins can be produced simultaneously, or serially. Thenext key advance in transient transfection technology is to approach orequal expression levels attained using stable expression systems withoutlosing the speed and flexibility of the transient format. We report forthe first time the development of a novel transient transfection systemthat utilizes high density 293F cell cultures to generate expressionlevels of >1 g/L (up to about 2 g/L) of human IgG and anon-IgG proteinswithin 7 days after cells are transfected.

To attain such high levels of protein expression, a novel cell culturesystem which includes a new high density growth culture medium incombination with a population of suspension cells that are adapted forhigh density growth in such a media was developed that allows certainpopulations of mammalian cells to reach viable cell densities of up to20×10⁶ cells/ml (more typically up to about 15×10⁶ cells/ml). Theseultra-high density cultures enable transfection at higher cell densitiesthan traditional protocols, significantly increasing the volumetricyield of protein. Additive The addition of one or more expressionenhancer formulations following or during transfection was also found toboost protein expression level to levels up to 10- to 12-fold higherthan the expression levels seen with current commercially availabletransient transfection systems. Parental suspension culture mammaliancells were adapted for improved growth and viability characteristicsunder high density culture conditions, and were then further selectedfor increased protein production. The resulting high density adaptedcells have an increased growth rate, increased cell size, and increasedspecific productivity compared to the parental cell line. Finally, thetransfection method was optimized through the use of one or moretransfections reagents that are used in combination with one or moreexpression enhancer formulations to further increase overall proteinyield.

When all of these improvements were combined into a single expressionsystem, protein levels were increased up to 10-fold for both IgG andnon-IgG recombinant proteins compared to the commercially availableFreeStyle™ 293 expression system and expression levels of >1 g/L wereattained for multiple proteins. Additionally, protein functionality wasdemonstrated to be comparable for several proteins expressed in the highyield expression system of the present invention when compared to thepopular commercially available FreeStyle™ 293 system. Together, theseresults indicate that significant increases in functional protein yieldscan be attained using a novel transient mammalian expression system thatincorporates numerous advances in protein expression technology into asingle, easy to use format.

The present invention also provides a method of cultivating eukaryoticcells comprising: (a) contacting the cells with the cell culture mediumof the present invention; (b) maintaining the cells under conditionssuitable to support cultivation of the cells in culture; and (c)optionally expressing a nucleic acid to form a protein product.

The present invention also provides a method for introducing one or moremacromolecules into at least one eukaryotic cell in culture, the methodcomprising: (a) culturing at least one eukaryotic cell in the medium ofclaim 1 in culture; (b) introducing at least one macromolecule into theculture under conditions sufficient to cause one or more of the at leastone macromolecule to be introduced in the at least one cell; and (c)cultivating the at least one cell in the medium to produce a productwhose production is controlled by the at least one molecule, whereingrowth of the at least one cell continues in the medium in the absenceof the medium being with fresh medium, wherein it is not necessary toremove medium used during the introduction from the presence of the atleast one cell to support growth of the at least one cell, and/orwherein after the introduction, growth is accomplished in cultivation ina volume of medium that is about the same volume up to no more thanabout 10 times the volume of the medium in which the introductionoccurred.

The present invention also provides a kit for the cultivation andtransfection of cells in vitro, the kit comprising the cell culturemedium of the present invention, and optionally further comprising oneor more of: one or more agents for the introduction of at least onemolecule into a cell, one or more macromolecules, at least one cell, andinstructions for culturing the at least one cell in culture and/or forintroducing at least one macromolecule into at least one cell inculture.

The present invention also provides a composition comprising the cellculture medium of the present invention and at least one componentselected from the group consisting of at least one eukaryotic cell, oneor more agents for the introduction of at least one macromolecule intoat least one cell, and one or more macromolecules.

Other embodiments of the present invention will be apparent to one ofordinary skill in light of the following drawings and description of theinvention, and of the claims.

BRIEF DESCRIPTION OF DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention.

In the drawings:

FIG. 1 is a graph demonstrating cell densities that are achievable usingthe transient transfection system in accordance with some embodiments ofthe invention. Cell adapted for high density growth were slowly adaptedinto various media over 3 passages. The media to which the cells wereadapted include High Density Culture Media in accordance with oneembodiment of the invention (closed circles), Test Media 1 (closedtriangles), Test Media 2 (open triangles), Test Media 3 (open diamonds).Cells were cultured for multiple passages in each of the media beforebeing seeded in 30 ml flasks at 0.2×10⁶ cells/ml. Cell density andviability were monitored over 8 days;

FIG. 2 is a bar graph outlining cell line expression optimization foruse with a transient transfection system in accordance with someembodiments of the invention. A parental 293F cell line was slit intomultiple subcultures which were subsequently adapted into a High DensityCulture Media. Various subcultures that were able to grown at highdensity were then selected and assessed for their ability to express arecombinant test protein (human IgG). The subculture of cells markedHigh Yield Adapted 293F Cells (right set of bars) expressed between 35%to 45% more recombinant IgG than two different subcultures of cellsderived from the same parental 293F cell line;

FIG. 3 is a bar graph outlining the effects of various enhancers used ina transient transfection system in accordance with some embodiments.Expression enhancers were identified that significantly improved proteinproduction. Components were formulated into 2 stable Enhancer solutions.The addition of Expression Enhancer 1 doubles hIgG expression (comparefirst two bars). The addition of Enhancer 2 by itself shows onlymarginal effect on enhancing expression of IgG, but when added incombination with Enhancer 1, provides almost 3 fold more hIgG vs.control (Compare third and fourth);

FIG. 4 shows a comparison of expression levels for 4 different andunique proteins using a high yield transient transfection system inaccordance with some embodiments and a prior art transient transfectionsystem (Freestyle™ 293 system). FIG. 4A shows a greater than 5-foldincrease in expression of human IgG using the transient transfectionsystem according to some embodiments of the present invention whencompared to commercially available FreeStyle™ Max system. FIG. 4B showsa greater than 5.2-fold increase in expression of Cripto using thetransient transfection system according to some embodiments of thepresent invention when compared to commercially available FreeStyle™ Maxsystem. FIG. 4C shows a almost 4-fold increase in expression of132-adrenergic receptor using the transient transfection systemaccording to some embodiments of the present invention when compared tocommercially available FreeStyle™ Max system. FIG. 4D shows a greaterthan 11-fold increase in expression of rabbit IgG using the transienttransfection system according to some embodiments of the presentinvention when compared to commercially available FreeStyle™ Max system;

FIG. 5 is a bar graph comparing the expression levels of EPO achievedusing a transient transfection system in accordance with someembodiments and a prior art transient transfection system. EPO wasexpressed using the transient expression system of the present inventionand Freestyle™ 293 system. The inventive system is scalable from 1 ml(24-well plate format) up to 1 L (3 L shake flask format). Reliablereproducibility in expression levels of specific proteins was seen inresults from three separate analysts in three different labs.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides improved medium formulations for thegrowth of both eukaryotic and prokaryotic cells. The inventive mediasupport cell growth, introduction of macromolecules into cells inculture and cell cultivation without requiring replenishment,replacement, supplementation, or changing medium between growth,introduction and/or cultivation. The media of the present invention canbe used to support or enhance the growth and cultivation of any cell.The present invention also provides compounds that can be used assubstitutes or to replace one or more undesired components, e.g., animalderived components. The replacement compounds provide at least onedesired function of the undesired component.

Definitions

In the description that follows, a number of terms used in cell cultureand recombinant DNA technology are utilized extensively. In order toprovide a clear and more consistent understanding of the specificationand claims, including the scope to be given such terms, the followingdefinitions are provided.

The term “introduction” of a macromolecule or compound into culturerefers to the provision of the macromolecule or compound into theculture medium.

The term “introduction” of a macromolecule or compound into at least onecell refers to the provision of a macromolecule or compound to a cell,such that the macromolecule or compound becomes internalized in thecell. For example, a macromolecule or compound can be introduced into acell using transfection, transformation, injection, and/or liposomalintroduction, and may also be introduced into a cell using other methodsknown to those of ordinary skill in the art. Preferably, a macromoleculeor compound is introduced into a cell by liposomal introduction. Themacromolecule is preferably a protein, peptide, polypeptide, or nucleicacid. The macromolecule may a protein. Alternatively, the macromoleculemay be a peptide. Alternatively, the macromolecule may be a polypeptide.The macromolecule may also be a nucleic acid.

The term “macromolecule,” as used herein, encompasses biomolecules. Inone embodiment, the term macromolecule refers to nucleic acid. In apreferred embodiment, the term macromolecule refers to deoxyribonucleicacid (DNA) and ribonucleic acid (RNA). More preferably, the termmacromolecule refers to DNA. More preferably, the term macromoleculerefers to complementary DNA (cDNA). A macromolecule can be charged oruncharged. A DNA molecule is an example of a charged macromolecule. Insome instances, the term “macromolecule”, as used herein, may be usedinterchangeably with the term “expressible nucleic acid”.

The term “transfection” is used herein to mean the delivery of nucleicacid, protein or other macromolecule to a target cell, such that thenucleic acid, protein or other macromolecule is expressed or has abiological function in the cell.

The term “expressible nucleic acid” as used herein includes both DNA andRNA without regard to molecular weight, and the term “expression” meansany manifestation of the functional presence of the nucleic acid withinthe cell including, without limitation, both transient expression andstable expression. Functional aspects include inhibition of expressionby oligonucleotides or protein delivery.

The term “expression of nucleic acid” and their equivalents refer to thereplication of the nucleic acid in a cell, to transcription of DNA tomessenger RNA, to translation of RNA to protein, to post-translationalmodification of protein, and/or to protein trafficking in the cell, orvariations or combinations thereof.

The term “ingredient” refers to any compound, whether of chemical orbiological origin, that can be used in cell culture media to maintain orpromote the growth or proliferation of cells. The terms “component,”“nutrient” and “ingredient” can be used interchangeably and are allmeant to refer to such compounds. Typical ingredients that are used incell culture media include amino acids, salts, metals, sugars, lipids,nucleic acids, hormones, vitamins, fatty acids, proteins and the like.Other ingredients that promote or maintain cultivation of cells ex vivocan be selected by those of skill in the art, in accordance with theparticular need. Media of the present invention can include one or morecomponents selected from the group consisting of bovine serum albumin(BSA) or human serum albumin (HSA), a one or more growth factors derivedfrom natural (animal) or recombinant sources such as epidermal growthfactor (EGF) or fibroblast growth factor (FGF), one or more lipids, suchas fatty acids, sterols and phospholipids, one or more lipid derivativesand complexes, such as phosphoethanolamine, ethanolamine andlipoproteins, one or more proteins, one or more and steroid hormones,such as insulin, hydrocortisone and progesterone, one or more nucleotideprecursors; and one or more trace elements.

The term “cell” as used herein refers includes all types of eukaryoticand prokaryotic cells. In preferred embodiments, the term refers toeukaryotic cells, especially mammalian cells. In certain exemplarythough non-limiting embodiments, the term “cell” is meant to refer tohuman 293 cells, or a variant thereof, such as, e.g., a 293 variant thatcan grow in suspension. Particularly preferred are variants of 293 cellsthat can grow, proliferate and be transfected in suspension culture, inparticular those variants that can be cultured at high density (e.g.,greater than about 2×10⁶ cells/ml, more preferably greater than about3×10⁶ cells/ml, or even optionally greater than about 4×10⁶ cells/ml).An example of such a variant 293 cell line is EXPI293™Fcells. In otherexemplary though non-limiting embodiments, the term “cell” is meant torefer to a CHO cell.

As used herein, the term “high density” when used in the context ofculturing cells in accordance with the present invention, and of methodsof the invention employing same for the purpose of conductingtransfection workflows, generally refers to a known cell line, or avariant of a known cell line, that can be grown or cultured in anappropriate cell culture medium to densities of greater than about 1×10⁶cells/ml, more preferably greater than about 2×10⁶ cells/ml, mostpreferably greater than about 3×10⁶ cells/ml, or even optionally greaterthan about 4×10⁶ cells/ml, or more up to about 20×10⁶ cells/ml, whilestill retaining the ability to be transfected at high efficiency and areable to express a target protein at high levels (e.g., levels exceeding200 g/ml to up to about 1 mg/ml or more.

The phrase “high density culture medium” is used herein to refer to anyculture medium capable of sustaining the growth of mammalian cells,preferably cells growing in suspension, at densities of up to about2×10⁷ cells/ml while maintaining viability of said cells in excess ofabout 80% and further, maintaining the ability of said suspension cellsto be efficiently transfected and express high amounts of recombinantprotein. The “high density culture medium” used in the practice of thepresent invention may vary between different applications and uses, andmay depend on the nature of the cell line being used, the desiredprotein being transiently expressed, the nature of the transfectionmodality selected for transfer of the expression vector into cells, andthe amount and nature of any expression enhancers added to the system asdescribed below. Nevertheless, preferred “high density culture medium”contemplated for use in the present transient expression systems andmethods will typically be serum-free, protein-free, allow thecultivation and growth of suspension cells to a density of up to about2×10⁷ cells/ml, more typically between about 2×10⁶ cells/ml to about1×10⁷ cells/ml, and will further enable the yield of protein produced inthe transient expression system to exceed at least 200 μg/mL of cellculture up to 2 mg/mL of cell culture, more typically between about 500μg/ml of cell culture to about 1 mg/mL of cell culture. Ideally, thehigh density culture medium used in accordance with the presentinvention will facilitate the transfection of cells at densities in therange of about 1×10⁶ to about 20×10⁶ cells/ml, about 2×10⁶ to about2×10⁶ cells/ml, or about 2.5×10⁶ to about 6×10⁶ cells/ml. Exemplary highdensity culture media suitable for use in the practice of the presentinvention include, though are not limited to, HuMEC Basal Serum freeMedium, KNOCKOUT™ CTS™ XenoFREE ESC/iPSC Medium, STEMPRO™-34 SFM Medium,STEMPRO™ NSC Medium, ESSENTIAL™-8 Medium, Medium 254, Medium, 106,Medium, 131, Medium, 154, Medium, 171, Medium 171, Medium 200, Medium231, HeptoZYME-SFM, Human Endothelial-SFM, GIBCO® FREESTYLE™ 293Expression Medium, Medium 154CF/PRF, Medium 154C, Medium 154 CF, Medium106, Medium 200PRF, Medium 131, Essential™-6 Medium, STEMPRO™-34 Medium,Gibco® Astrocyte Medium, AIM V® Medium CTS™, AMINOMAX™ C-100 BasalMedium, AMINOMAX™-II Complete Medium, CD FORTICHO™ Medium, CD CHO AGTMedium, CHO-S-SFM Medium, GIBCO®FREESTYLE™ CHO Expression Medium, CDOPTICHO™ Medium, CD CHO Medium, CD DG44 Medium, SF-900™ Medium, EXPI293™Expression Medium, LHC Basal Medium, LHC-8 Medium, 293 SFM Medium, CD293 Medium, AEM Growth Medium, PER. C6® Cell Medium, AIM V® Medium,EXPILIFE® Medium, Keratinocyte-SFM Medium, LHC Medium, LHC-8 Medium,LHC-9 Medium, and any derivatives or modifications thereof. In certainpreferred though non-limiting embodiments, a high density culture mediamay be CD FORTICHO™ Medium, CD CHO AGT Medium, CHO-S-SFM Medium,GIBCO®FREESTYLE™ CHO Expression Medium, CD OPTICHO™ Medium, CD CHOMedium, CD DG44 Medium, GIBCO® FREESTYLE™ 293 Expression Medium,EXPI293™ Expression Medium, or a like medium, or a modified versionthereof. The above listed exemplary high density culture media may beparticularly suitable for the high density growth, propagation,transfection and maintenance of CHO cells, a CHO cell variant, 293cells, a 293 cell variant, CapT cells, a CapT cell variant, or any othercells adapted for use in a high density culture system.

The phrase “cells adapted for high density culture” is meant to refer toa cell lineage or a (non-clonal) population of cells derived from thesame parental cell lineage that has been adapted to grow at high densityin a high-density culture medium while retaining cell viability at orabove about 80%. Such cells may be isolated or selected out from theparental population of cells by maintaining the cells at high densityover >40, >50, >60, >70, or >80 sequential passages and graduallyreplacing the proportion of growth medium with the desired high-densityculture medium. Optionally, during the process, different pools of cellsmay be individually propagated and subjected to the selection procedurewhile simultaneously assessing transfection efficiency and or proteinexpression efficiency, so that non-clonal population of cells may beselected that can be sustained and grown at high density, transfectedwith high efficiency, and express high levels of a desired recombinantprotein. While it will be readily apparent to the skilled practitionerthat a variety of cell types and lineages may be subjected to thisselection procedure, it has been determined that cell lineages derivedfrom CHO cells, cell lineages derived from 293 fibroblast cells, andcells derived from CapT cells are particularly amenable to the selectionprocess for being adapted to high density growth conditions. Ideally,cells that are adapted to high density growth culture and amenable foruse in the present invention will also be capable of being transfectedat high efficiency and/or capable of expressing recombinant protein atyield exceeding at least 200 about μg/mL of cell culture up to about 2mg/mL of cell culture, more typically between about 500 μg/ml of cellculture to about 1 mg/mL of cell culture. Ideally, cells adapted forhigh density culture used in accordance with the present invention arecapable of being sustained and transfected at densities in the range ofabout 1×10⁶ to about 20×10⁶ cells/ml, about 2×10⁶ to about 2×10⁶cells/ml, or about 2.5×10⁶ to about 6×10⁶ cells/ml.

By “cell culture” or “culture” is meant the maintenance of cells in anartificial, in vitro environment.

By “cultivation” is meant the maintenance of cells in vitro underconditions favoring growth and/or differentiation and/or or continuedviability. “Cultivation” can be used interchangeably with “cellculture.” Cultivation is assessed by number of viable cells/ml culturemedium. Cultivation after introduction of a macromolecule preferablyincludes production of a product, for example, a protein product on avirus.

The term “replenishing, replacing, or supplementing medium” as usedherein refers to adding a volume of fresh cell culture medium to mediumthat was already present in culture and/or replacing medium that wasalready present in culture with fresh medium, and/or supplementingmedium already present in culture with new medium. Fresh medium ismedium that does not contain the one or more macromolecules or compoundsto be introduced into at least one cell or medium that has not been incontact with cells to support their growth on cultivation. The skilledartisan can determine whether there is an advantage from or a need toremove and/or replenish, replace or supplement medium by monitoring cellgrowth and/or viability by techniques known in the art, such as cellcounting (manual or automated), trypan blue exclusion, production ofprotein or other substance, alamar blue assay, presence or concentrationof one or more metabolic products, cell adhesion, morphologicalappearance, analysis of spent medium, etc. One or a combination ofmonitoring techniques can be used to determine whether the medium needsto be to support growth, introduction of at least one macromoleculeand/or cultivation after introduction of at least one macromolecule.

“Recombinant protein” refers to protein that is encoded by a nucleicacid that is introduced into a host cell. The host cell expresses thenucleic acid. The term “expressing a nucleic acid” is synonymous with“expressing a protein from an RNA encoded by a nucleic acid. “Protein”as used herein broadly refers to polymerized amino acids, e.g.,peptides, polypeptides, proteins, lipoproteins, glycoproteins, etc.

The term “protein yield” refers to the amount of protein expressed bycultured cells, and can be measured, for example, in terms of grams ofprotein produced/ml medium. If the protein is not secreted by the cells,the protein can be isolated from the interior of the cells by methodsknown to those of ordinary skill in the art. If the protein is secretedby the cells, the protein can be isolated from the culture medium bymethods known to those of ordinary skill in the art. The amount ofprotein expressed by the cell can readily be determined by those ofordinary skill in the art. The protein may be a recombinant protein.

A “protein product” is a product associated with production or an actionby a protein. A protein product may be a protein. A protein product mayalso be a product resulting from action of a protein by one or moreother substances to produce a product. An example of such action isenzymatic action by a protein.

By “suspension culture” is meant cell culture in which the majority orall of cells in a culture vessel are present in suspension, and theminority or none of the cells in the culture vessel are attached to thevessel surface or to another surface within the vessel. Preferably,“suspension culture” has greater than 75% of the cells in the culturevessel are in suspension, not attached to a surface on or in the culturevessel. More preferably, a “suspension culture” has greater than 85% ofthe cells in the culture vessel are present in suspension, not attachedto a surface on or in the culture vessel. Even more preferred is a“suspension culture” with greater than 95% of the cells in the culturevessel present in suspension, not attached to a surface on or in theculture vessel.

The medium, methods, kit and composition of the present invention aresuitable for either monolayer or suspension culture, transfection, andcultivation of cells, and for expression of protein in cells inmonolayer or suspension culture. Preferably, the medium, methods, kitand composition of the present invention are for suspension culture,transfection, and cultivation of cells, and for expression of proteinproduct in cells in suspension culture.

By “culture vessel” is meant any container, for example, a glass,plastic, or metal container, that can provide an aseptic environment forculturing cells.

The phrases “cell culture medium,” “tissue culture medium,” “culturemedium” (plural “media” in each case) and “medium formulation” refer toa nutritive solution for cultivating cells or tissues. These phrases canbe used interchangeably.

The term “combining” refers to the mixing or admixing of ingredients.

Derivative of a molecule includes some compounds that comprise the basemolecule, but have additional or modified side groups. Preferably, a“derivative” can be formed by reacting the base molecule with only 1,but possibly 2, 3, 4, 5, 6, etc. reactant molecules. A single stepreaction is preferred, but multi-step, e.g., 2, 3, 4, 5, 6, etc.reactions are known in the art to form derivatives. Substitution,condensation and hydrolysis reactions are preferred and may be combinedto form the derivative compound. Alternatively, a derivative compoundmay be a compound that preferably in 1, but possibly 2, 3, 4, 5, 6, etc.reactions can form the base compound or a substitution or condensationproduct thereto.

A cell culture medium is composed of a number of ingredients and theseingredients can vary from medium to medium. Each ingredient used in acell culture medium has its unique physical and chemicalcharacteristics. Compatibility and stability of ingredients aredetermined in part by the “solubility” of the ingredients in aqueoussolution. The terms “solubility” and “soluble” refer to the ability ofan ingredient to form and remain in solution with other ingredients.Ingredients are thus compatible if they can be maintained in solutionwithout forming a measurable or detectable precipitate.

By “compatible ingredients” is also meant those media components whichcan be maintained together in solution and form a “stable” combination.A solution containing “compatible ingredients” is said to be “stable”when the ingredients do not precipitate, degrade or decomposesubstantially such that the concentration of one or more of thecomponents available to the cells from the media is reduced to a levelthat no longer supports the optimum or desired growth of the cells.Ingredients are also considered “stable” if degradation cannot bedetected or when degradation occurs at a slower rate when compared todecomposition of the same ingredient in a 1× cell culture mediaformulation. For example, in 1× media formulations glutamine is known todegrade into pyrolidone carboxylic acid and ammonia. Glutamine incombination with divalent cations are considered “compatibleingredients” since little or no decomposition of the glutamine can bedetected over time in solutions or combinations in which both glutamineand divalent cations are present. See U.S. Pat. No. 5,474,931. Thus, theterm “compatible ingredients” as used herein refers to the combinationof particular culture media ingredients which, when mixed in solutioneither as concentrated or 1× formulations, are “stable” and “soluble.”

The term “1× formulation” is meant to refer to any aqueous solution thatcontains some or all ingredients found in a cell culture medium atworking concentrations. The “1× formulation” can refer to, for example,the cell culture medium or to any subgroup of ingredients for thatmedium. The concentration of an ingredient in a 1× solution is about thesame as the concentration of that ingredient found in a cell cultureformulation used for maintaining or cultivating cells in vitro. A cellculture medium used for the in vitro cultivation of cells is a 1×formulation by definition. When a number of ingredients are present,each ingredient in a 1× formulation has a concentration about equal tothe concentration of each respective ingredient in a medium during cellculturing. For example, RPMI-1640 culture medium contains, among otheringredients, 0.2 g/L L-arginine, 0.05 g/L L-asparagine, and 0.02 g/LL-aspartic acid. A “1× formulation” of these amino acids contains aboutthe same concentrations of these ingredients in solution. Thus, whenreferring to a “1× formulation,” it is intended that each ingredient insolution has the same or about the same concentration as that found inthe cell culture medium being described. The concentrations ofingredients in a 1× formulation of cell culture medium are well known tothose of ordinary skill in the art. See, for example, Methods ForPreparation of Media, Supplements and Substrate For Serum-Free AnimalCell Culture Allen R. Liss, N.Y. (1984), Handbook of MicrobiologicalMedia, Second Ed., Ronald M. Atlas, ed. Lawrence C. Parks (1997) CRCPress, Boca Raton, Fla. and Plant Culture Media, Vol. 1: Formulationsand Uses E. F. George, D. J. M. Puttock, and H. J. George (1987)Exegetics Ltd. Edington, Westbury, Wilts, BA13 4QG England each of whichis incorporated by reference herein in its entirety. The osmolarityand/or pH, however, can differ in a 1× formulation compared to theculture medium, particularly when fewer ingredients are contained in the1× formulation.

A “10× formulation” is meant to refer to a solution wherein theconcentration of each ingredient in that solution is about 10 times morethan the concentration of each respective ingredient in a medium duringcell culturing. For example, a 10× formulation of RPMI-1640 culturemedium can contain, among other ingredients, 2.0 g/L L-arginine, 0.5 g/LL-asparagine, and 0.2 g/L L-aspartic acid (compare 1× formulation,above). A “10× formulation” can contain a number of additionalingredients at a concentration about 10 times that found in the 1×culture formulation. As will be readily apparent, “25× formulation,”“50× formulation,” “100× formulation,” “500× formulation,” and “1000×formulation” designate solutions that contain ingredients at about 25-,50-, 100-, 500-, or 1000-fold concentrations, respectively, as comparedto a 1× cell culture formulation. Again, the osmolarity and pH of themedium formulation and concentrated solution can vary.

The term “trace element” or “trace element moiety” refers to a moietywhich is present in a cell culture medium in only very low (i.e.,“trace”) amounts or concentrations, relative to the amounts orconcentrations of other moieties or components present in the culturemedium. In the present invention, these terms encompass Ag⁺, Al³⁺, Ba²⁺,Cd²⁺, Co²⁺, Cr³⁺, Cu¹⁺, Cu²⁺, Fe²⁺, Fe³⁺, Ge⁴⁺, Se⁴⁺, Br⁻, I⁻, Mn²⁺, F⁻,Si⁴⁺, V⁵⁺, Mo⁶⁺, Ni²⁺, Rb⁺, Sn²⁺ and Zr⁴⁺ and salts thereof. Forexample, the following salts can be used as trace elements in theculture media of the invention: AgNO₃, AlCl₃.6H₂O, Ba(C₂H₃O₂)₂,CdSO₄.8H₂O, CoCl₂.6H₂O, Cr₂(SO₄)₃.1H₂O, GeO₂, Na₂SeO₃, H₂SeO₃, KBr, KI,MnCl₂.4H₂O, NaF, Na₂SiO₃.9H₂O, NaVO₃, (NH₄)₆Mo₇O₂₄.4H₂O, NiSO₄.6H₂O,RbCl, SnCl₂, and ZrOCl₂.8H₂O. Suitable concentrations of trace elementmoieties can be determined by one of ordinary skill in the art usingonly routine experimentation.

The term “amino acid” refers to amino acids or their derivatives (e.g.,amino acid analogs), as well as their D- and L-forms. Examples of suchamino acids include glycine, L-alanine, L-asparagine, L-cysteine,L-aspartic acid, L-glutamic acid, L-phenylalanine, L-histidine,L-isoleucine, L-lysine, L-leucine, L-glutamine, L-arginine,L-methionine, L-proline, L-hydroxyproline, L-serine, L-threonine,L-tryptophan, L-tyrosine, and L-valine, N-acetyl cysteine.

A “chemically defined” medium is one in which each chemical species andits respective quantity is known prior to its use in culturing cells. Achemically defined medium is made without lysates or hydrolysates whosechemical species are not known and/or quantified. A chemically definedmedium is one preferred embodiment of the medium of the presentinvention.

The terms “serum-free culture conditions” and “serum-free conditions”refer to cell culture conditions that exclude serum of any type. Theseterms can be used interchangeably.

A “serum-free medium” (sometimes referred to as “SFM Medium”) is amedium that contains no serum (e.g., fetal bovine serum (FBS), calfserum, horse serum, goat serum, human serum, etc.) and is generallydesignated by the letters SFM. Exemplary though non-limiting serum-freemedia familiar to the skilled artisan include HuMEC Basal Serum freeMedium, KNOCKOUT™ CTS™ XenoFREE ESC/iPSC Medium, STEMPRO™-34 SFM Medium,STEMPRO™ NSC Medium, ESSENTIAL™-8 Medium, Medium 254, Medium, 106,Medium, 131, Medium, 154, Medium, 171, Medium 171, Medium 200, Medium231, HeptoZYME-SFM, Human Endothelial-SFM, GIBCO® FREESTYLE™ 293Expression Medium, Medium 154CF/PRF, Medium 154C, Medium 154 CF, Medium106, Medium 200PRF, Medium 131, Essential™-6 Medium, STEMPRO™-34 Medium,Gibco® Astrocyte Medium, AIM V® Medium CTS™, AMINOMAX™ C-100 BasalMedium, AMINOMAX™-II Complete Medium, CD FORTICHO™ Medium, CD CHO AGTMedium, CHO-S-SFM Medium, GIBCO®FREESTYLE™ CHO Expression Medium, CDOPTICHO™ Medium, CD CHO Medium, CD DG44 Medium, SF-900™ Medium, EXPI293™Expression Medium, LHC Basal Medium, LHC-8 Medium, 293 SFM Medium, CD293 Medium, AEM Growth Medium, PER. C6® Cell Medium, AIM V® Medium,EXPILIFE® Medium, Keratinocyte-SFM Medium, LHC Medium, LHC-8 Medium,LHC-9 Medium, and any derivatives or modifications thereof.

The phrase “protein-free” culture media refers to culture media thatcontain no protein (e.g., no serum proteins such as serum albumin orattachment factors, nutritive proteins such as growth factors, or metalion carrier proteins such as transferrin, ceruloplasmin, etc.).Preferably, if peptides are present, the peptides are smaller peptides,e.g., di- or tri-peptides. Preferably, peptides of deca-peptide lengthor greater are less than about 1%, more preferably less than about 0.1%,and even more preferably less than about 0.01% of the amino acidspresent in the protein free medium.

The phrase “low-protein” culture media as used herein refers to mediathat contain only low amounts of protein (typically less than about 10%,less than about 5%, less than about 1%, less than about 0.5%, or lessthan about 0.1%, of the amount or concentration of total protein foundin culture media containing standard amounts of protein, such asstandard basal medium supplemented with 5-10% serum).

The term “animal derived” material as used herein refers to materialthat is derived in whole or in part from an animal source, includingrecombinant animal DNA or recombinant animal protein DNA. Preferredmedia contain no animal desired material.

The term “expression enhancer” generally refers to one or more liquid(preferably aqueous) additives used to supplement a culture mediumformulation in accordance with the presently described embodiments, saidadditives being selected to improve the yield of expressed proteinproduced in a transient protein expression system in accordance with thepresently described embodiments. The term encompasses any one or more ofseveral compounds that affect cell cycle progression, inhibit apoptosis,slow cell growth and/or promote protein production. In the context ofthe present invention, the term “expression enhancers” generally refersto any one or more compounds added to a transient transfection system,the presence of which enhances or promotes expression of a targetprotein by a factor of at least 2 fold up to about 10-fold above theexpression level seen in the absence of such expression enhancer(s).Exemplary expression enhancers suitable for use with the presentlydescribed embodiments include, though are not limited to, additives suchas valproic acid (VPA, acid and sodium salt), sodium propionate, lithiumacetate, dimethyl sulfoxide (DMASO), sugars including galactose, aminoacid mixtures, or butyric acid, or any combinations of theaforementioned. The optimal concentration of each specific expressionenhancer may vary according to individual characteristics of theexpression system and the requirements of the user, and thedetermination of what constitutes an optimal concentration of any one ormore expression enhancer in a given experimental scenario is well withinpurview of a practitioner having ordinary skill level in the art. By wayof example only, in some embodiments, the optimal final concentrationsranges of valproic acid (VPA) used in the practice of the presentinvention may be in the range of about 0.20 mM to about 25 mM. Morepreferably, the final concentration of VPA may be in the range of about0.25 mM to about 24 mM, about 0.26 mM to about 23 mM, 0.27 mM to about23 mM, 0.28 mM to about 23 mM, 0.29 mM to about 22 mM, about 0.30 mM toabout 21 mM, about 0.31 mM to about 20 mM, about 0.32 mM to about 19 mM,about 0.33 mM to about 17 mM, about 0.34 mM to about 18 mM, about 0.35mM to about 17 mM, about 0.36 mM to about 16 mM, about 0.37 mM to about15 mM, about 0.40 mM to about 14 mM, about 0.41 mM to about 13 mM, about0.42 mM to about 12 mM, about 0.43 mM to about 11 mM, about 0.44 mM toabout 10 mM, about 0.45 mM to about 9 mM, about 0.46 mM to about 8 mM,about 0.47 mM to about 7 mM, about 0.48 mM to about 6 mM, about 0.49 mMto about 5 mM, about 0.50 mM to about 4 mM, about 0.50 mM to about 4 mM,about 0.55 mM to about 3 mM, 0.6 mM to about 2 mM or 0.75 to about 1.5mM. In some preferred though non-limiting embodiments, the finalconcentration of VPA used in the practice of the present invention maybe between about 0.15 mM to about 1.5 mM, about 0.16 mM to about 1.5 mM,about 0.17 mM to about 1.5 mM, about 0.18 mM to about 1.5 mM, about 0.19mM to about 1.5 mM, about 0.20 mM to about 1.5 mM, about 0.25 mM toabout 1.5 mM, about 0.30 mM to about 1.5 mM, about 0.40 mM to about 1.5mM, about 0.50 mM to about 1.5 mM, about 0.60 mM to about 1.5 mM, about0.70 mM to about 1.5 mM, about 0.80 mM to about 1.5 mM, about 0.90 mM toabout 1.5 mM or about 0.10 mM to about 1.5 mM. In some preferred thoughnon-limiting embodiments, the final concentration of VPA used in thepractice of the present invention may be between about 0.20 to about 1.5mM, about 0.21 to about 1.4 mM, about 0.22 to about 1.4 mM, about 0.23to about 1.4 mM, about 0.24 to about 1.4 mM, about 0.25 to about 1.3 mM,about 0.25 to about 1.2 mM, about 0.25 to about 1.1 mM, or about 0.25 toabout 1.0 mM.

In further embodiments, the optimal final concentration of sodiumpropionate (NaPP) used in the practice of the present invention may bein the range of about 0.2 mM to about 100 mM. In certain preferredthough non-limiting embodiments, the optimal final concentration of NAPPmay be in the range of about 0.5 to about 80 mM, about 0.4 mM to about70 mM, about 0.5 mM to about 60 mM, about 0.6 mM to about 50 mM, about0.7 mM to about 40 mM, about 0.8 mM to about 30 mM, about 0.9 mM toabout 20 mM, about 1 mM to about 15 mM, about 2 mM to about 10 mM, about3 mM to about 9 mM, about 4 mM to about 8 mM, or about 5 mM to about 7mM. In certain preferred though non-limiting embodiments, the optimalfinal concentration of NAPP may be in the range of about 1 mM to about10 mM, about 1 mM to about 2 mM, about 2 mM to about 3 mM, about 3 mM toabout 4 mM, about 4 mM to about 5 mM, about 5 mM to about 6 mM, about 6mM to about 7 mM, about 7 mM to about 8 mM, about 8 mM to about 9 mM, orabout 9 mM to about 10 mM. In certain preferred though non-limitingembodiments, the optimal final concentration of NAPP may be about 1 mM,about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM,about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, about9.5 mM, or about 10 mM.

In further embodiments, the optimal final concentration of lithiumacetate (LiAc) used in the practice of the present invention may be inthe range of about 0.25 to about 25 mM, about 0.26 mM to about 20 mM,about 0.27 mM to about 15 mM, about 0.28 mM to about 10 mM, about 0.29mM to about 5 mM, about 0.3 mM to about 4.5 mM, about 0.31 mM to about 4mM, about 0.35 mM to about 3 mM, about 0.5 mM to about 2.5 mM, about 1mM to about 3 mM, about 1.5 mM to about 2.5 mM, or about 2 mM to about 3mM.

In further embodiments, the optimal final concentration of butyric acidused in the practice of the present invention may be in the range ofabout 0.25 to about 25 mM, about 0.26 mM to about 20 mM, about 0.27 mMto about 15 mM, about 0.28 mM to about 10 mM, about 0.29 mM to about 5mM, about 0.3 mM to about 4.5 mM, about 0.31 mM to about 4 mM, about0.35 mM to about 3 mM, about 0.5 mM to about 2.5 mM, about 1 mM to about3 mM, about 1.5 mM to about 2.5 mM, or about 2 mM to about 3 mM.

An expression enhancer used in accordance with the present invention maybe added to the culture medium immediately prior to transfection orafter transfection prior to harvesting the cells and the expressedprotein. In some specific though non-limiting embodiments describedbelow, “Enhancer 1” generally refers to 0.25 mM-1 mM valproic acid, and“Enhancer 2” generally refers to 5 mM-7 mM sodium propionate. However,if indicated otherwise, the terms Enhancer 1 and Enhancer 2 mayencompass different enhancer compounds. Expression enhancers may beadded to a culture medium sequentially, or as a cocktail.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA into which additional DNA segments may beligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors,” or simply, “expressionvectors.” In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector. Certain vectors usedin accordance with the practice of invention described herein may bewell-known vectors used in the art, such as, e.g., pCDNA 3.3, or amodified version thereof. Non-limiting examples of the types ofmodification to a vector that may be suitable in the practice of thepresent invention include, though are not limited to, modification suchas the addition of modification of one or more enhancers, one or morepromoters, one or more ribosomal binding sites, one or more origins ofreplication, or the like. In certain preferred though non-limitingembodiments, and expression vector used in the practice of the presentinvention may include one or more enhancer elements selected to improveexpression of the protein of interest in the present transientexpression system. The selected enhancer element may be positioned 5′ or3′ to the expressible nucleic acid sequence used to express the proteinof interest. A particularly preferred though non-limiting enhancerelement is the woodchuck hepatitis post-transcriptional regulatoryelement (WPRE).

As used herein, the phrase “expression vector containing a geneticsequence capable of producing an expressed protein” generally refers toa vector as defined above which is capable to accommodating anexpressible nucleic acid sequence having at least one open-reading frameof a desired protein of interest (said protein of interest beingselected by the user of the present invention) in additional to one ormore nucleic acid sequences or elements that are required to support theexpression thereof in a cell or in a cell-free expression system. Suchadditional nucleic acid sequences or elements that may be present in anexpression vector as defined herein may include, one or more promotersequences, one or more enhancer elements, one or more ribosomal bindingsites, one or more translational initiation sequences, one or moreorigins of replication, or one or more selectable markers. A variety ofnucleic acid sequences or elements serving this purpose are familiar tothe skilled artisan, and the selection of one or more thereof for use inthe practice of the present invention is well within the purview of theskilled practitioner.

The terms “polynucleotide” and “nucleic acid” as used herein refers toany nucleic acid, including deoxyribonucleic acid (DNA) and ribonucleicacid (RNA). In preferred embodiments, “nucleic acid” refers to DNA,including genomic DNA, complementary DNA (cDNA), and oligonucleotides,including oligo DNA. In certain preferred though non-limitingembodiments, “nucleic acid’ refers to genomic DNA and/or cDNA. Thenucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase or by a syntheticreaction. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may comprise modification(s)made after synthesis, such as conjugation to a label. Other types ofmodifications include, for example, “caps,” substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotides(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars may be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or may be conjugated to solid or semi-solidsupports. The 5′ and 3′ terminal OH can be phosphorylated or substitutedwith amines or organic capping group moieties of from 1 to 20 carbonatoms. Other hydroxyls may also be derivatized to standard protectinggroups. Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl-, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such asarabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs, and basic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), (O)NR₂ (“amidate”), P(O)R,P(O)OR′, CO, or CH2 (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

“Oligonucleotide,” as used herein, generally refers to short, generallysingle-stranded, generally synthetic polynucleotides that are generally,but not necessarily, less than about 200 nucleotides in length. Theterms “oligonucleotide” and “polynucleotide” are not mutually exclusive.The description above for polynucleotides is equally and fullyapplicable to oligonucleotides.

As used herein, the phrase “first period of time”, when used in thecontext of a method for transiently transfecting cells in accordancewith the methods of the invention described herein generally refers tothe time interval between transfecting a population of cells with anexpressible nucleic acid and the additional of one or more expressionenhancers to the transfected cells. Typically, a first period of timewill be in the range of about 2 hrs to about 4 days. In certainpreferred though non-limiting embodiments, a first period of time may bein the range of about 3 to about 90 hrs, about 4 to about 85 hr, about 5to about 80 hrs, about 6 to about 75 hrs, about 7 to about 70 hrs, about8 to about 65 hrs, about 9 to about 60 hrs, about 10 to about 55 hrs,about 11 to about 50 hrs, about 12 to about 45 hrs, about 13 to about 40hrs, about 14 to about 35 hrs, about 15 to 30 hrs, about 16 to about 24hrs, about 17 to about 24 hrs, about 18 to about 24 hrs, about 19 toabout 24 hrs, about 20 to about 24 hrs, about 21 to about 24 hrs, about22 to about 24 hrs or about 23 to about 24 hrs. In other preferred tonon-limiting embodiments, a first period of time may be up to about 15hrs, up to about 16 hrs, up to about 17 hrs, up to about 18 hrs, up toabout 19 hrs, up to about 20 hrs, up to about 21 hrs, up to about 22hrs, up to about 23 hrs, up to about 24 hrs, up to about 25 hrs, up toabout 26 hrs, up to about 27 hrs, up to about 28 hrs, up to about 29 hrsor up to about 30 hrs.

As used herein, the phrase “second period of time”, when used in thecontext of a method for transiently transfecting cells in accordancewith the methods of the invention described herein generally refers tothe time interval between the addition of one or more expressionenhancers and either the addition of one or more additional enhancers,or the harvesting of the transfected cells and purification or isolationof the protein expressed therein. Typically, a second period of timewill be in the range of about 10 hrs to about 10 days, though other timeintervals may be used if determined to be optimal for the protein beingexpressed. In some preferred though non-limiting embodiments, the secondperiod of time may be in the range of 2 hrs to 5 days, 2.5 hrs to 4days, about 3 to about 90 hrs, about 4 to about 85 hr, about 5 to about80 hrs, about 6 to about 75 hrs, about 7 to about 70 hrs, about 8 toabout 65 hrs, about 9 to about 60 hrs, about 10 to about 55 hrs, about11 to about 50 hrs, about 12 to about 45 hrs, about 13 to about 40 hrs,about 14 to about 35 hrs, about 15 to 30 hrs, about 16 to about 24 hrs,about 17 to about 24 hrs, about 18 to about 24 hrs, about 19 to about 24hrs, about 20 to about 24 hrs, about 21 to about 24 hrs, about 22 toabout 24 hrs or about 23 to about 24 hrs. In other preferred tonon-limiting embodiments, a first period of time may be up to about 15hrs, up to about 16 hrs, up to about 17 hrs, up to about 18 hrs, up toabout 19 hrs, up to about 20 hrs, up to about 21 hrs, up to about 22hrs, up to about 23 hrs, up to about 24 hrs, up to about 25 hrs, up toabout 26 hrs, up to about 27 hrs, up to about 28 hrs, up to about 29 hrsor up to about 30 hrs.

As used herein the phrase “third period of time”, when used in thecontext of a method for transiently transfecting cells in accordancewith the methods of the invention described herein generally refers tothe time interval between the addition of at least a first expressionenhancer and at least a second expression enhancer. The time intervalbetween the addition of a first and second expression enhancer may be onthe order of seconds to days, though in some embodiments such first andsecond expression enhancer may be added essentially simultaneous, or mayoptionally be provided in a single formulation.

As used herein the terms “complexation reaction,” “complexation media”or the like, generally refer to a physiologically acceptable culturemedia or reaction in which a nucleic acid is complexed to a transfectionreagent formulation. Typically, a nucleic acid that is to be introducedinto a cell for the purpose of expressing a protein is first complexedwith a suitable transfection reagent (such as, e.g., a cationic lipidformulation) to lipid/nucleic acid complexes or aggregates.

By “transition element” or “transition metal” (which can be usedinterchangeably) is meant an element in which an inner electron valenceshell, rather than an outer shell, is only partially filled, such thatthe element acts as a transitional link between the most and leastelectropositive in a given series of elements. Transition elements aretypically characterized by high melting points, high densities, highdipole or magnetic moments, multiple valencies, and the ability to formstable complex ions. Examples of such transition elements useful in thepresent invention include scandium (Sc), titanium (Ti), vanadium (V),chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni),copper (Cu), zinc (Zn), yttrium (Y), zirconium (Zr), niobium (Nb),molybdenum (Mo), technetium (Tc), rubidium (Ru), rhodium (Rh), palladium(Pd), silver (Ag), cadmium (Cd), lanthanum (La), hafnium (Hf), tantalum(Ta), tungsten (W), rhenium (Re), osmium (Os), iridium (Ir), platinum(Pt), gold (Au), mercury (Hg), and actinium (Ac). Of particular interestas a transition element for use in culture media compositions, includingthose of the present invention, are ions, chelates, salts, and complexesof iron (Fe²⁺ or Fe³⁺).

A variety of techniques and reagents are available for the introductionof macromolecules into a target cell in a process known as“transfection”. Commonly used reagents include, for example, calciumphosphate, DEAE-dextran and lipids. For examples of detailed protocolsfor the use of reagents of these types, numerous references texts areavailable for example, Current Protocols in Molecular Biology, Chapter9, Ausubel, et al. Eds., John Wiley and Sons, 1998. Additional methodsfor transfecting cells are known in the art, and may includeelectroporation (gene electrotransfer), sono-poration, opticaltransfection, protoplast fusion, impalefection, magnetofection, or viraltransduction.

A “reagent for the introduction of macromolecules” into cells or a“transfection reagent” is any material, formulation or composition knownto those of skill in the art that facilitates the entry of amacromolecule into a cell. For example, see U.S. Pat. No. 5,279,833. Insome embodiments, the reagent can be a “transfection reagent” and can beany compound and/or composition that increases the uptake of one or morenucleic acids into one or more target cells. A variety of transfectionreagents are known to those skilled in the art. Suitable transfectionreagents can include, but are not limited to, one or more compoundsand/or compositions comprising cationic polymers such aspolyethyleneimine (PEI), polymers of positively charged amino acids suchas polylysine and polyarginine, positively charged dendrimers andfractured dendrimers, cationic β-cyclodextrin containing polymers(CD-polymers), DEAE-dextran and the like. In some embodiments, a reagentfor the introduction of macromolecules into cells can comprise one ormore lipids which can be cationic lipids and/or neutral lipids.Preferred lipids include, but are not limited to,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylamonium chloride (DOTMA),dioleoylphosphatidylcholine (DOPE),1,2-Bis(oleoyloxy)-3-(4′-trimethylammonio) propane (DOTAP),1,2-dioleoyl-3-(4′-trimethylammonio) butanoyl-sn-glycerol (DOTB),1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC), cholesteryl(4′-trimethylammonio)butanoate (ChoTB), cetyltrimethylammonium bromide(CTAB), 1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide (DORI),1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE),1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide(DMRIE),O,O′-didodecyl-N-[p(2-trimethylammonioethyloxy)benzoyl]-N,N,N-trimethylam-moniumchloride, spermine conjugated to one or more lipids (for example,5-carboxyspermylglycine dioctadecylamide (DOGS),N,N^(I),N^(II),N^(III)-tetramethyl-N,N^(I),N^(II),N^(III)-tet-rapalmitylspermine(TM-TPS) and dipalmitoylphasphatidylethanolamine 5-carboxyspermylaminde(DPPES)), lipopolylysine (polylysine conjugated to DOPE), TRIS(Tris(hydroxymethyl)aminomethane, tromethamine) conjugated fatty acids(TFAs) and/or peptides such as trilysyl-alanyl-TRIS mono-, di-, andtri-palmitate, (3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol(DC-Chol), N-(α-trimethylammonioacetyl)-didodecyl-D-glutamate chloride(TMAG), dimethyl dioctadecylammonium bromide (DDAB),2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanamin-iniumtrifluoroacetate(DOSPA) and combinations thereof.

Those skilled in the art will appreciate that certain combinations ofthe above mentioned lipids have been shown to be particularly suited forthe introduction of nucleic acids into cells for example a 3:1 (w/w)combination of DOSPA and DOPE is available from Life TechnologiesCorporation, Carlsbad, Calif. under the trade name LIPOFECTAMINE™, a 1:1(w/w) combination of DOTMA and DOPE is available from Life TechnologiesCorporation, Carlsbad, Calif. under the trade name LIPOFECTIN®, a 1:1(M/M) combination of DMRIE and cholesterol is available from LifeTechnologies Corporation, Carlsbad, Calif. under the trade name DMRIE-Creagent, a 1:1.5 (M/M) combination of TM-TPS and DOPE is available fromLife Technologies Corporation, Carlsbad, Calif. under the trade nameCellFECTIN® and a 1:2.5 (w/w) combination of DDAB and DOPE is availablefrom Life Technologies Corporation, Carlsbad, Calif. under the tradename LipfectACE®. In addition to the above-mentioned lipid combinations,other formulations comprising lipids in admixture with other compounds,in particular, in admixture with peptides and proteins comprisingnuclear localization sequences, are known to those skilled in the art.For example, see international application no. PCT/US99/26825, publishedas WO 00/27795, both of which are incorporated by reference herein.

Lipid aggregates such as liposomes have been found to be useful asagents for the delivery of macromolecules into cells. In particular,lipid aggregates comprising one or more cationic lipids have beendemonstrated to be extremely efficient at the delivery of anionicmacromolecules (for example, nucleic acids) into cells. One commonlyused cationic lipid isN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).Liposomes comprising DOTMA alone or as a 1:1 mixture withdioleoylphosphatidylethanolamine (DOPE) have been used to introducenucleic acids into cells. A 1:1 mixture of DOTMA:DOPE is commerciallyavailable from Life Technologies Corporation, Carlsbad, Calif. under thetrade name of LIPOFECTIN™. Another cationic lipid that has been used tointroduce nucleic acids into cells is1,2-bis(oleoyl-oxy)-3-3-(trimethylammonia) propane (DOTAP). DOTAPdiffers from DOTMA in that the oleoyl moieties are linked to thepropylamine backbone via ether bonds in DOTAP whereas they are linkedvia ester bonds in DOTMA. DOTAP is believed to be more readily degradedby the target cells. A structurally related group of compounds whereinone of the methyl groups of the trimethylammonium moiety is replacedwith a hydroxyethyl group are similar in structure to the Rosenthalinhibitor (RI) of phospholipase A (see Rosenthal, et al., (1960) J.Biol. Chem. 233:2202-2206.). The RI has stearoyl esters linked to thepropylamine core. The dioleoyl analogs of RI are commonly abbreviatedDOR1-ether and DOR1-ester, depending upon the linkage of the lipidmoiety to the propylamine core. The hydroxyl group of the hydroxyethylmoiety can be further derivatized, for example, by esterification tocarboxyspermine.

Another class of compounds which has been used for the introduction ofmacromolecules into cells comprise a carboxyspermine moiety attached toa lipid (see, Behr, et al., (1989) Proceedings of the National Academyof Sciences, USA 86:6982-6986 and EPO 0 394 111). Examples of compoundsof this type include dipalmitoylphosphatidylethanolamine5-carboxyspermylamide (DPPES) and 5-carboxyspermylglycinedioctadecylamide (DOGS). DOGS is commercially available from Promega,Madison, Wis. under the trade name of TRANSFECTAM™.

A cationic derivative of cholesterol(3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol, DC-Chol) hasbeen synthesized and formulated into liposomes with DOPE (see Gao, etal., (1991) BBRC 179(1):280-285.) and used to introduce DNA into cells.The liposomes thus formulated were reported to efficiently introduce DNAinto the cells with a low level of cellular toxicity. Lipopolylysine,formed by conjugating polylysine to DOPE (see Zhou, et al., (1991) BBA1065:8-14), has been reported to be effective at introducing nucleicacids into cells in the presence of serum.

Other types of cationic lipids that have been used to introduce nucleicacids into cells include highly packed polycationic ammonium, sulfoniumand phosphonium lipids such as those described in U.S. Pat. Nos.5,674,908 and 5,834,439, and international application no.PCT/US99/26825, published as WO 00/27795. One particularly preferredthough non-limiting transfection reagent for delivery of macromoleculesin accordance with the present invention is LIPOFECTAMINE 2000™ which isavailable from Life technologies (see U.S. international application no.PCT/US99/26825, published as WO 00/27795). Another preferred thoughnon-limiting transfection reagent suitable for delivery ofmacromolecules to a cell is EXPIFECTAMINE™. Other suitable transfectionreagents include LIOFECTAMINE™ RNAiMAX, LIPOFECTAMINE™ LTX,OLIGOFECTAMINE™, Cellfectin™, INVIVOFECTAMINE™, INVIVOFECTAMINE™ 2.0,and any of the lipid reagents or formulations disclosed in U.S. PatentAppl. Pub. No. 2012/0136073, by Yang et al. (incorporated herein byreference thereto). A variety of other transfection reagents are knownto the skilled artisan and may be evaluated for the suitability thereofto the transient transfection systems and methods described herein.

The present invention is directed to a high-yield transient transfectionsystem that supports (a) the introduction of at least one macromolecule,preferably an expressible nucleic acid molecule, into eukaryotic cellsin culture, (b) the cultivation of cells into which at least onemacromolecule is introduced, and optionally (c) the production ofrecombinant protein product or expression of the nucleic acid in cellsinto which at least one macromolecule is introduced, wherein mediumcontaining the macromolecule does not need to be removed from theculture and replaced with fresh medium after introduction of at leastone macromolecule into cells and prior to cultivation and production ofprotein product or expression of nucleic acid.

The transient transfection system of the present invention, an the usethereof in accordance with the methods described herein, results in therapid and reproducible expression of high levels of a protein ofinterest in a cell culture system. Typically, the present transienttransfection systems and methods are capable of producing recombinantexpressed protein at levels in the range of about 200 μg protein/L ofculture to about 2 g protein/L of culture, depending on the individualexpression characteristics of the desired recombinant protein and celltype used. Using the transient transfection system and methods providedfor herein, a user may obtain levels of expressed protein that are about2-fold to up to about 20-fold in excess of what is currently obtainableusing standard commercially available transient transfection systems.Using the transient transfection system and methods provided for herein,a user may obtain levels of expressed protein that is about 2.5-fold,about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, bout 7-fold, about7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, orup to about 10-fold or greater than that seen with contemporarytransient expression systems. For example, using the present transienttransfection system to produce a recombinant protein, a user may obtaina protein yield between about 2-fold up to about 10-fold higher than theprotein yield obtained using a commercially available transienttransfection system optimized for production of recombinant protein insuspension cells, such as, e.g., FREESTYLE™ Expression System.

Using the system of the present invention, which system includes, amongother elements, at least a high density culture medium, at least apopulation of suspension cells adapted for high density growth,optionally one or more expression vectors, optionally one or moretransfection reagents, and optionally one or more expression enhancers,it is not necessary to replenish, replace or supplement the medium afterone has introduced at least one macromolecule into at least one cell,and before cells into which at least one macromolecule has beenintroduced are further cultured to produces protein product or express anucleic acid. In the system of the present invention, the medium isideally a serum-free medium and/or a chemically defined medium and/orprotein free or substantially low protein medium, and/or a medium thatdoes not contain animal derived components, or a medium havingcombinations of these features.

In one non-limiting aspect of the invention, with respect to theintroduction of compounds or macromolecules (e.g., nucleic acid) intocells in culture, the high yield culture medium of the present inventionfacilitates higher cell transfection efficiency than can typically beobtained using presently available transient transfection systems. Inanother related though non-limiting aspect of the invention, the systemalso does not require transfecting the cells in a smaller volume thancells are to be cultured in after transfection. In yet another relatedthough non-limiting aspect of the present invention, the systemfacilitates higher cell viability than presently available transienttransfection systems. In yet a further related though non-limitingaspect still, the system facilitates higher cell density (i.e., cells/mlof culture medium) than presently available transient transfectionsystems. In another related though non-limiting aspect of the presentinvention, the system facilitates a higher level of recombinant proteinexpression in cells in culture than presently available transienttransfection systems. Preferably, though not necessarily, the samevolume of medium can be used for to introduce at least one macromoleculeinto a cell and subsequent cultivation without having to replace,remove, supplement or replenish the medium in which the transfection ofthe cells has occurred. Alternatively, the cells are divided or mediumvolume is increased less from about 2, about 5, about 8 or about 10times.

The medium, methods, kit and composition of the present invention areintended to be used to introduce at least one macromolecule or totransfect and culture cells in any volume of culture medium. Suchintroduction is preferably accomplished in 0.1 to 10 times the amount ofmedium used to culture cells to be transfected. Preferably, the cellculture volume is greater than about one milliliter. More preferably,the cell culture volume is from about 200 μl to 100 liters. Morepreferably, the cell culture volume is from about 2 ml to about 50liters, most preferably from about 5 ml to about 5 liters. Morepreferably, the cell culture volume is from about 100 ml to about 50liters. More preferably, the cell culture volume is from about 500 ml toabout 50 liters. More preferably, the cell culture volume is from about500 ml to about 25 liters. More preferably, the cell culture volume isfrom about 500 ml to about 10 liters. More preferably, the cell culturevolume is from about 500 ml to about 5 liters. More preferably, the cellculture volume is from about 500 ml to about 1 liter.

In the medium, methods, kit and composition of the present invention,the medium optionally does not contain compounds that can interfere withintroduction of macromolecules or transfection, e.g., polyanioniccompounds such as polysulfonated and/or polysulfated compounds.Preferably, the medium does not contain dextran sulfate.

The medium, methods, kit and composition of the present invention permitthe introduction of compounds or macromolecules (particularlymacromolecules, for example nucleic acids, proteins and peptides) intothe cultured cells (for example by transfection) without the need tochange the medium. In one preferred embodiment, the present inventionprovides a medium for the cultivation and transfection of eukaryoticcells.

Using the medium, methods, kit and composition of the present invention,those of ordinary skill in the art can introduce macromolecules orcompounds (e.g., nucleic acid) into cells in culture. Preferably, themacromolecule or compound (e.g., nucleic acid) is introduced into atleast about 20 percent of the cells. More preferably, the macromoleculeor compound (e.g., nucleic acid) is introduced into about 20 to about100 percent of the cells. More preferably, the macromolecule or compound(e.g., nucleic acid) is introduced into about 30 to about 100 percent ofthe cells. More preferably, the macromolecule or compound (e.g., nucleicacid) is introduced into about 50 to about 100 percent of the cells.Practically, the macromolecule or compound might be introduced intoabout 20% to about 90% of the cells, about 20% to about 80% of thecells, about 30% to about 60, 70, 80 or 90% of the cells, about 20, 30,40 or 50% to about 70, 75, 80, 85, 90, 95 or 98% of the cells, etc. Evenabout 60, 70, 75 or 80 to about 90% or close to 100% of the cells maycontain the introduced molecule or compound.

In preferred embodiments of the medium, methods, kit and composition ofthe present invention, one or more undesirable components (i.e., one ormore serum components, one or more undefined components, one or moreprotein components and/or one or more animal derived components) havebeen substituted or replaced in one or more functions by one or morereplacement compounds. Replacement compounds of the invention mayoptionally include one or more metal binding compounds and/or one ormore transition element complexes, said complexes comprising one or moretransition elements or a salts or ions thereof, in a complex with one ormore metal-binding compounds. Preferably, the medium is capable ofsupporting the cultivation of a cell in vitro in the absence of one ormore naturally derived metal carriers, such as transferrin, or otheranimal derived proteins or extracts. The metal binding compound can bein a complex with a transition metal prior to addition of the metalbinding compound to the medium. In other embodiments, the metal bindingcompound is not in a complex with a transition metal prior to additionof the metal binding compound to the media. Preferably, the medium ofthe present invention does not contain transferrin and/or does notcontain insulin.

The present invention also relates to a cell culture medium obtained bycombining a medium with one or more replacement compounds. Preferably,the medium can be a serum-free medium and/or a chemically defined mediumand/or a protein-free or low protein medium and/or can be a mediumlacking animal derived components. The medium preferably does notcontain transferrin and/or does not contain insulin. In some preferredembodiments, the medium can be capable of supporting the cultivation ofa cell in vitro and/or can permit the introduction of macromoleculesinto the cell. In some embodiments, one or more of the replacementcompounds can be a metal binding compound and/or can be a transitionelement complex, said complex comprising at least one transition elementor a salt or ion thereof complexed to at least one metal-bindingcompound. Preferred transition elements, metal-binding compounds, andtransition element complexes for use in this aspect of the inventioninclude those described in detail herein.

Replacement compounds of the present invention can facilitate thedelivery of transition metals to cells cultured in vitro. In preferredembodiments, the replacement compounds can deliver iron and replacetransferrin. A preferred replacement compound is a hydroxypyridinederivative. Preferably, the hydroxypyridine derivative is selected fromthe group consisting of 2-hydroxypyridine-N-oxide, 3-hydroxy-4-pyrone,3-hydroxypypyrid-2-one, 3-hydroxypyrid-2-one, 3-hydroxypyrid-4-one,1-hydroxypyrid-2-one, 1,2-dimethyl-3-hydroxypyrid-4-one,1-methyl-3-hydroxypyrid-2-one, 3-hydroxy-2(1H)-pyridinone, and pyridoxalisonicotinyl hydrazone, nicotinic acid-N-oxide, 2-hydroxy-nicotinicacid. Most preferably, the hydroxypyridine derivative is2-hydroxypyridine-N-oxide.

The replacement compounds of the present invention can be used with anymedia, including media for cultivating or growing eukaryotic and/orprokaryotic cells, tissues, organs, etc. Such media include, but are notlimited to, CD FORTICHO™ Medium, Expi293™ Expression Media, Dulbecco'sModified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), BasalMedium Eagle (BME), RPMI-1640, Ham's F-10, Ham's F-12, aMinimalEssential Medium (aMEM), Glasgow's Minimal Essential Medium (G-MEM), andIscove's Modified Dulbecco's Medium (IMDM). Other media that arecommercially available (e.g., from Life Technologies Corporation,Carlsbad, Calif.) or that are otherwise known in the art can beequivalently used in accordance with the present invention including,but not limited to, 293 SFM, CD-CHO medium, VP SFM, BGJb medium,Brinster's BMOC-3 medium, cell culture freezing medium, CMRL media, EHAAmedium, eRDF medium, Fischer's medium, Gamborg's B-5 medium, GLUTAMAX™supplemented media, Grace's insect cell media, HEPES buffered media,Richter's modified MEM, IPL-41 insect cell medium, Leibovitz's L-15media, McCoy's 5A media, MCDB 131 medium, Media 199, Modified Eagle'sMedium (MEM), Medium NCTC-109, Schneider's Drosophila medium, TC-100insect medium, Waymouth's MB 752/1 media, William's Media E, proteinfree hybridoma medium II (PFHM II), AIM V media, Keratinocyte SFM,defined Keratinocyte SFM, STEMPRO® SFM, STEMPRO® completemethylcellulose medium, HepatoZYME-SFM, Neurobasal™ medium, Neurobasal-Amedium, Hibernate™ A medium, Hibernate E medium, Endothelial SFM, HumanEndothelial SFM, Hybridoma SFM, PFHM II, Sf 900 medium, Sf 900 TI SFM,EXPRESS FIVE® medium, CHO-S-SFM, AMINOMAX-II complete medium,AMINOMAX-C100 complete medium, AMINOMAX-C 100 basal medium, PB-MAX™karyotyping medium, KARYOMAX bone marrow karyotyping medium, KNOCKOUTD-MEM and CO₂ independent medium. The above media are obtained frommanufacturers known to those of ordinary skill in the art, such as JRH,Sigma, HyClone, and BioWhittaker. Additional examples of media suitablefor use in the practice of the present invention can be found in U.S.Pat. Nos. 5,135,866 and 5,232,848 as well as in internationalpublications nos. WO 88/02774, WO 98/15614, WO 98/08934 and EuropeanPatent No. 0 282 942, the entireties of which are specificallyincorporated herein by reference.

The present invention also provides a method for introducingmacromolecules into cells, comprising culturing cells in a medium of theinvention and contacting the cells in the medium with one or moremacromolecules under conditions causing the macromolecules to be takenup by one or more of the cells. Preferably, the medium is a serum-freemedium and/or a chemically defined medium and/or a protein-free or lowprotein medium and/or can be a medium lacking animal derived components.Preferred cells include eukaryotic cells. More preferably, the cells aremammalian cells. The medium can comprise one or more replacementcompounds and preferably does not contain transferrin and/or does notcontain insulin. In some preferred embodiments, the medium permits thegrowth and transfection of the cell in the same medium. In someembodiments, the macromolecules can comprise one or more nucleic acidsand conditions causing the nucleic acid molecules to be taken up by thecells include contacting the nucleic acid with a reagent which causesthe nucleic acid to be introduced into one or more cells.

The present invention also provides a composition comprising a medium ofthe invention and a cell. Preferably, the medium is a serum-free mediumand/or a chemically defined medium and/or a protein-free or low proteinmedium and/or a medium lacking animal derived components. Preferredcells include eukaryotic cells. More preferably, the cells are mammaliancells. Most preferred are suspension cells derived from 293 fibroblasts.The medium can comprise one or more replacement compounds and preferablydoes not contain transferrin and/or does not contain insulin.Preferably, the medium supports the growth and transfection of the cellin the same medium, more preferably, the medium supports the growth andcultivation of mammalian cells expressing a recombinant protein, wheresaid medium does not have to be replenished, replaced or otherwisesupplemented after the introduction of an expressible nucleic acidtherein for the purposes of producing a recombinant protein.

The present invention also provides compositions comprising a medium ofthe present invention and one or more reagents for the introduction ofmacromolecules into one or more cells. Preferably, the medium is aserum-free medium and/or a chemically defined medium and/or aprotein-free or low protein medium and/or a medium lacking animalderived components. The medium can comprise one or more replacementcompounds and preferably does not contain transferrin and/or does notcontain insulin. Preferably, the medium contains a transfection reagentand the macromolecules are nucleic acids. The macromolecules might alsobe proteins and/or peptides. In some embodiments, the reagent comprisesone or more lipids of which one or more can be cationic lipids. Morepreferably, the reagent comprises a mixture of neutral and cationiclipids. In some embodiments, the reagent comprises one or more peptidesand/or proteins which can be provided alone or in admixture with one ormore lipids.

The present invention also provides compositions comprising a medium ofthe invention and one or more macromolecules to be introduced into acell. Preferably, the medium is a serum-free medium and/or a chemicallydefined medium and/or a protein-free or low protein medium and/or amedium lacking animal derived components. The medium can comprise one ormore replacement compounds and preferably does not contain transferrinand/or does not contain insulin. The macromolecules can be, for example,nucleic acids and/or proteins and/or peptides and can be uncomplexed orcan be in the form of a complex with one or more reagents for theintroduction of macromolecules into cells. Preferably, themacromolecules are nucleic acids and can be in the form of a complexwith one or more transfection reagents.

The present invention also provides a composition comprising at leastone component (or combination thereof) selected from the groupconsisting of a medium of the present invention, at least one cell, atleast one macromolecule, at least one reagent for introducing at leastone macromolecule into at least one cell. Preferably, the cells areeukaryotic cells. More preferably, the cells are mammalian cells.Preferably, the medium is a serum-free medium and/or a chemicallydefined medium and/or a protein-free or low protein medium and/or amedium lacking animal derived components. The medium can comprise one ormore replacement compounds and preferably does not contain transferrinand/or does not contain insulin. In some preferred embodiments, thereagent is a transfection reagent and the macromolecules are nucleicacids, for example RNA and/or DNA. Alternatively, the macromolecules areproteins and/or peptides.

In some embodiments, the reagent comprises one or more lipids of whichone or more can be cationic lipids. More preferably, the reagentcomprises a mixture of neutral and cationic lipids. In some embodiments,the reagent comprises one or more peptides and/or proteins which can beprovided alone or in admixture with one or more lipids. In preferredembodiments, the reagent complexes with the macromolecule to introducethe macromolecule into the cell.

The present invention also provides kits for the culture andtransfection of cells comprising at least one container comprising amedium for the culture and transfection of cells. Such kits may alsocomprise at least one component (or a combination thereof) selected fromthe group consisting of a medium of the present invention, at least onecell, at least one macromolecule, at least one reagent for introducingat least one macromolecule into at least one cell, at least one bufferor buffering salt, and instructions for using the kit to introduce atleast one macromolecule into at least one cell. Preferably, the mediumis a serum-free medium and/or a chemically defined medium and/or aprotein-free or low protein medium and/or a medium lacking animalderived components. The medium can comprise one or more replacementcompounds and preferably does not contain transferrin and/or does notcontain insulin and/or does not contain an animal growth factor. Themedium can comprise one or more replacement compounds that can be metalbinding compounds and/or can comprise one or more complexes comprisingone or more replacement compounds. In some embodiments, the medium cancomprise one or more complexes, said complex comprising one or moretransition elements or salts or ions thereof complexed one or morereplacement compounds which can be metal-binding compounds. In someembodiments, said medium is capable of supporting the cultivation of acell in vitro and permits transfection of cells cultured therein. Insome embodiments, kits of the invention can further comprise at leastone container comprising a lipid for transfecting cells. In someembodiments, the kits of the invention can comprise at least onecontainer comprising a nucleic acid.

According to one aspect of the invention, a transition element ispreferably selected from the group consisting of scandium, titanium,vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc,yttrium, zirconium, niobium, molybdenum, technetium, rubidium, rhodium,palladium, silver, cadmium, lanthanum, hafnium, tantalum, tungsten,rhenium, osmium, iridium, platinum, gold, mercury, and actinium, orsalts or ions thereof, and is preferably an iron salt. Suitable ironsalts include, but are not limited to, FeCl₃, Fe(NO₃) 3 or FeSO₄ orother compounds that contain Fe⁺⁺⁺ or Fe⁺⁺ ions.

Preferred replacement compounds include, but are not limited to,metal-binding compounds. See, for example, international patentapplication no. PCT/US00/23580, Publication No. WO 01/16294.

Metal binding compounds of the present invention include anymacromolecules which can interact with or bind with transition elementsand facilitate their uptake by cells. Such interaction/binding can becovalent or non-covalent in nature. The metal-binding compound used inthis aspect of the invention is preferably selected from the groupconsisting of a polyol, a hydroxypyridine derivative,1,3,5-N,N′,N″-tris(2,3-dihydroxybenzoyl)amino-methylbenzene,ethylenediamine-N,N′-tetramethylenephosphonic acid, trisuccin, an acidicsaccharide (e.g., ferrous gluconate), a glycosaminoglycan,diethylenetriaminepentaacetic acid, nicotinic acid-N-oxide,2-hydroxy-nicotinic acid, mono-, bis-, or tris-substituted2,2′-bipyridine, a hydroxamate derivative (e.g. acetohydroxamic acid),an amino acid derivative, deferoxamine, ferrioxamine, iron basicporphine and derivatives thereof, DOTA-lysine, a texaphyrin, asapphyrin, a polyaminocarboxylic acid, an α-hydroxycarboxylic acid, apolyethylenecarbamate, ethyl maltol, 3-hydroxy-2-pyridine, and IRCO11.In one preferred embodiment, the metal-binding compound is a polyol suchas sorbitol or dextran, and particularly sorbitol. In a relatedembodiment, the metal-binding compound is a hydroxypyridine derivative,such as 2-hydroxypyridine-N-oxide, 3-hydroxy-4-pyrone,3-hydroxypypyrid-2-one, 3-hydroxypyrid-2-one, 3-hydroxypyrid-4-one,1-hydroxypyrid-2-one, 1,2-dimethyl-3-hydroxypyrid-4-one,1-methyl-3-hydroxypyrid-2-one, 3-hydroxy-2(1H)-pyridinone, ethyl maltolor pyridoxal isonicotinyl hydrazone, and is preferably2-hydroxypyridine-N-oxide. In particularly preferred embodimentsaccording to this aspect of the invention, the transition metal complexcan be a sorbitol-iron complex or 2-hydroxypyridine-N-oxide-ironcomplex. The metal binding compounds of the present invention can alsobind divalent cations such as Ca⁺⁺ and Mg⁺⁺.

The invention relates to cell culture media comprising one or morereplacement compounds which can be metal-binding compounds and furthercomprising one or more ingredients selected from the group ofingredients consisting of at least one amino acid (such as L-alanine,L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid,L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine,L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine,L-tryptophan, L-tyrosine or L-valine, N-acetyl-cysteine), at least onevitamin (such as biotin, choline chloride, D-Ca⁺⁺-pantothenate, folicacid, i-inositol, niacinamide, pyridoxine, riboflavin, thiamine orvitamin B12), at least one inorganic salt (such as a calcium salt,CuSO₄, FeSO₄, Fe(NO₃)₃, FeCl₃, KCl, a magnesium salt, a manganese salt,sodium acetate, NaCl, NaHCO₃, Na₂HPO₄, Na.₂SO₄, a selenium salt, asilicon salt, a molybdenum salt, a vanadium salt, a nickel salt, a tinsalt, ZnCl₂, ZnSO₄ or other zinc salts), adenine, ethanolamine,D-glucose, one or more cytokines, heparin, hydrocortisone, lipoic acid,phenol red, phosphoethanolamine, putrescine, sodium pyruvate,tri-iodothyronine, PLURONIC F68, and thymidine.

The culture media of the present invention can optionally include one ormore buffering agents. Suitable buffering agents include, but are notlimited to, N-[2-hydroxyethyl]-piperazine-N′-[2-ethanesulfonic acid](HEPES), MOPS, MES, phosphate, bicarbonate and other buffering agentssuitable for use in cell culture applications. A suitable bufferingagent is one that provides buffering capacity without substantialcytotoxicity to the cells cultured. The selection of suitable bufferingagents is within the ambit of ordinary skill in the art of cell culture.

According to the invention, a medium suitable for use in forming thecell culture media of the invention can comprise one or moreingredients, and can be obtained, for example, by combining one or moreingredients selected from the group consisting of adenine, ethanolamine,D-glucose, heparin, a buffering agent, hydrocortisone, lipoic acid,phenol red, phosphoethanolamine, putrescine, sodium pyruvate,tri-iodothyronine, thymidine, L-alanine, L-arginine, L-asparagine,L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine,L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan,L-tyrosine, L-valine, N-acetyl-cysteine, biotin, choline chloride,D-Ca⁺⁺-pantothenate, folic acid, i-inositol, niacinamide, pyridoxine,riboflavin, thiamine, vitamin B12, Pluronic F68, recombinant insulin, acalcium salt, CuSO₄, FeSO₄, FeCl₃, Fe(NO₃)₃, KCl, a magnesium salt, amanganese salt, sodium acetate, NaCl, NaHCO₃, Na₂HPO₄, Na₂SO₄, aselenium salt, a silicon salt, a molybdenum salt, a vanadium salt, anickel salt, a tin salt, ZnCl₂, ZnSO₄ or other zinc salts, wherein eachingredient is added in an amount which supports the cultivation of acell in vitro.

The invention is also directed to a cell culture medium comprisingingredients selected from ethanolamine, D-glucose, HEPES, insulin,linoleic acid, lipoic acid, phenol red, PLURONIC F68, putrescine, sodiumpyruvate, transferrin, L-alanine, L-arginine, L-asparagine, L-asparticacid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine,L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine,L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine,biotin, choline chloride, D-Ca⁺⁺-pantothenate, folic acid, i-inositol,niacinamide, pyridoxine, riboflavin, thiamine, vitamin B12, one or morecalcium salts, Fe(NO₃)₃, KCl, one or more magnesium salts, one or moremanganese salts, NaCl, NaHCO₃, Na₂HPO₄, one or more selenium salts, oneor more vanadium salts and one or more zinc salts, wherein eachingredient is present in an amount which supports the suspensioncultivation of a mammalian epithelial cell in vitro. The invention isalso directed to such media which can optionally further comprise one ormore supplements selected from the group consisting of one or morecytokines, heparin, one or more animal peptides, one or more yeastpeptides and one or more plant peptides (most preferably one or more ofrice, aloevera, soy, maize, wheat, pea, squash, spinach, carrot, potato,sweet potato, tapioca, avocado, barley, coconut and/or green bean,and/or one or more other plants), e.g., see international applicationno. PCT/US97/18255, published as WO 98/15614.

The media provided by the present invention can be protein-free, and canbe a 1× formulation or concentrated as, for example, a 10×, 20×, 25×,50×, 10×, 500×, or 1000× medium formulation.

The media of the invention can also be prepared in different forms, suchas dry powder media (“DPM”), a granulated preparation (which requiresaddition of water, but not other processing, such as adjusting pH),liquid media or as media concentrates.

The basal medium that is a medium useful only for maintenance, but notfor growth or production of product, can comprise a number ofingredients, including amino acids, vitamins, organic and inorganicsalts, sugars and other components, each ingredient being present in anamount which supports the cultivation of a mammalian epithelial cell invitro.

In the medium, methods, kit and composition of the present invention,the medium can be used to culture a variety of cells. Preferably, themedium is used to culture eukaryotic cells. More preferably, the mediumis used to culture plant and/or animal cells. More preferably, themedium is used to culture mammalian cells, fish cells, insect cells,amphibian cells or avian cells. More preferably, the medium is used toculture mammalian cells. More preferably, the medium may be used toculture mammalian cells, including primary epithelial cells (e.g.,keratinocytes, cervical epithelial cells, bronchial epithelial cells,tracheal epithelial cells, kidney epithelial cells and retinalepithelial cells) and established cell lines and their strains (e.g.,293 embryonic kidney cells, BHK cells, HeLa cervical epithelial cellsand PER-C6 retinal cells, MDBK (NBL-1) cells, 911 cells, CRFK cells,MDCK cells, CapT cells, CHO cells, BeWo cells, Chang cells, Detroit 562cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LS180cells, LS174T cells, NCI-H-548 cells, RPMI 2650 cells, SW-13 cells, T24cells, WI-28 VA13, 2RA cells, WISH cells, BS-C-I cells, LLC-MK₂ cells,Clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Y-1 cells, LLC-PK₁cells, PK(15) cells, GH₁ cells, GH₃ cells, L2 cells, LLC-RC 256 cells,MH₁C₁ cells, XC cells, MDOK cells, VSW cells, and TH-I, B1 cells, orderivatives thereof), fibroblast cells from any tissue or organ(including but not limited to heart, liver, kidney, colon, intestines,esophagus, stomach, neural tissue (brain, spinal cord), lung, vasculartissue (artery, vein, capillary), lymphoid tissue (lymph gland, adenoid,tonsil, bone marrow, and blood), spleen, and fibroblast andfibroblast-like cell lines (e.g., CHO cells, TRG-2 cells, IMR-33 cells,Don cells, GHK-21 cells, citrullinemia cells, Dempsey cells, Detroit 551cells, Detroit 510 cells, Detroit 525 cells, Detroit 529 cells, Detroit532 cells, Detroit 539 cells, Detroit 548 cells, Detroit 573 cells, HEL299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, MiCl₁cells, CHO cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells,Vero cells, DBS-FrhL-2 cells, BALB/3T3 cells, F9 cells, SV-T2 cells,M-MSV-BALB/3T3 cells, K-BALB cells, BLO-11 cells, NOR-10 cells,C₃H/IOTI/2 cells, HSDM₁C₃ cells, KLN₂O₅ cells, McCoy cells, Mouse Lcells, Strain 2071 (Mouse L) cells, L-M strain (Mouse L) cells, L-MTK⁻(Mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss/3T3cells, Indian muntjac cells, SIRC cells, C_(II) cells, and Jensen cells,or derivatives thereof). Most preferably, the medium is used to culturemammalian cells selected from the group consisting of 293 cells, 293 Fcells or derivatives thereof, PER-C6 cells or derivatives thereof, CHOcells or derivatives thereof, CapT cells or derivatives thereof, COS-7Lcells or derivatives thereof and Sp2/0 cells or derivatives thereof, orany other suspension cell line or derivative capable of being culturedat high cell density as defined above. More preferably, the medium isused to culture 293 cells or a modified 293 cell line specificallyadapted for optimal growth in the cell culture medium that forms thebasis of the present invention. In some preferred though non-limitingaspects, the medium is used to culture cells in suspension.

Cells supported by the medium of the present invention can be derivedfrom any animal, preferably a mammal, and most preferably a mouse or ahuman. The cells cultivated in the present media can be normal cells orabnormal cells (i.e., transformed cells, established cells, or cellsderived from diseased tissue samples).

The present invention also provides methods of cultivating mammalianepithelial or fibroblast cells using the culture medium formulationsdisclosed herein, comprising (a) contacting the cells with the cellculture media of the invention; and (b) cultivating the cells underconditions suitable to support cultivation of the cells. In someembodiments, the methods of the present invention can optionally includea step of contacting the cultured cells with a solution comprising oneor more macromolecules (preferably comprising one or more nucleic acids)under conditions causing the introduction of one or more of themacromolecules into one or more of the cells. Preferably, cellscultivated according to these methods (which can include any of thecells described above) are cultivated in suspension.

In some aspects, a transient transfection and recombinant protein systemmay include a high density culture medium suitable for the growth andpropagation of cultured mammalian cells at densities in the range ofabout 1×10⁶ to about 20×10⁶ cells/ml, more preferably in the range ofabout 2×10⁶ to about 6×10⁶. Any culture medium may be used in thepractice of the present invention, with the proviso that the culturemedium employed is capable of sustaining the growth of mammalian cells,preferably cells growing in suspension, at densities of up to about2×10⁷ cells/ml while maintaining viability of said cells in excess ofabout 80% and further, maintaining the ability of said suspension cellsto be efficiently transfected and express high amounts of recombinantprotein. The high density culture medium used in the practice of thepresent invention may vary between different applications and uses, andmay depend on the nature of the cell line being used, the desiredprotein being transiently expressed, the nature of the transfectionmodality selected for transfer of the expression vector into cells, andthe amount and nature of any expression enhancers added to the system asdescribed below. Nevertheless, preferred high density culture mediumcontemplated for use in the present transient expression systems andmethods will typically be serum-free, protein-free, allow thecultivation and growth of suspension cells to a density of up to about2×10⁷ cells/ml, more typically between about 2×10⁶ cells/ml to about1×10⁷ cells/ml, and will further enable the yield of protein produced inthe transient expression system to exceed at least 200 μg/mL of cellculture up to 2 mg/mL of cell culture, more typically between about 500μg/ml of cell culture to about 1 mg/mL of cell culture. Ideally, thehigh density culture medium used in accordance with the presentinvention will facilitate the transfection of cells at densities in therange of about 1×10⁶ to about 20×10⁶ cells/ml, about 2×10⁶ to about2×10⁶ cells/ml, or about 2.5×10⁶ to about 6×10⁶ cells/ml.

Particularly preferred high density growth media suitable for thepractice of the present invention may be a chemically defined medium inwhich each chemical species and its respective quantity is known priorto its use in culturing cells. The selected chemically defined mediummay optionally be made without cellular or tissue lysates orhydrolysates whose chemical species are not known and/or quantified.

In some aspects of the present invention a particularly suited type ofmedium for the practice of the present invention is a serum-free medium(sometimes referred to as “SFM Medium”) being entirely devoid of, e.g.,fetal bovine serum (FBS), calf serum, horse serum, goat serum, humanserum, and the like. Exemplary though non-limiting serum-free mediafamiliar to the skilled artisan include HuMEC Basal Serum free Medium,KNOCKOUT™ CTS™ XenoFREE ESC/iPSC Medium, STEMPRO™-34 SFM Medium,STEMPRO™ NSC Medium, ESSENTIAL™-8 Medium, Medium 254, Medium, 106,Medium, 131, Medium, 154, Medium, 171, Medium 171, Medium 200, Medium231, HeptoZYME-SFM, Human Endothelial-SFM, GIBCO® FREESTYLE™ 293Expression Medium, Medium 154CF/PRF, Medium 154C, Medium 154 CF, Medium106, Medium 200PRF, Medium 131, Essential™-6 Medium, STEMPRO™-34 Medium,Gibco® Astrocyte Medium, AIM V® Medium CTS™, AMINOMAX™ C-100 BasalMedium, AMINOMAX™-II Complete Medium, CD FORTICHO™ Medium, CD CHO AGTMedium, CHO-S-SFM Medium, GIBCO®FREESTYLE™ CHO Expression Medium, CDOPTICHO™ Medium, CD CHO Medium, CD DG44 Medium, SF-900™ Medium, EXPI293™Expression Medium, LHC Basal Medium, LHC-8 Medium, 293 SFM Medium, CD293 Medium, AEM Growth Medium, PER. C6® Cell Medium, AIM V® Medium,EXPILIFE® Medium, Keratinocyte-SFM Medium, LHC Medium, LHC-8 Medium,LHC-9 Medium, and any derivatives or modifications thereof.

In some aspects of the present invention a particularly suited type ofmedium for the practice of the present invention is a protein-freemedium (sometimes referred to as “PFM Medium”) being entirely devoid ofprotein (e.g., no serum proteins such as serum albumin or attachmentfactors, nutritive proteins such as growth factors, or metal ion carrierproteins such as transferrin, ceruloplasmin, etc.). Preferably, ifpeptides are present, the peptides are smaller peptides, e.g., di- ortri-peptides. Preferably, peptides of deca-peptide length or greater areless than about 1%, more preferably less than about 0.1%, and even morepreferably less than about 0.01% of the amino acids present in theprotein free medium.

Ideally, both serum-free and protein-free media contemplated for usewith the present invention will further be devoid of any animal derivedmaterial, or any material that is derived in whole or in part from ananimal source, including recombinant animal DNA or recombinant animalprotein DNA.

Exemplary high density culture media suitable for use in the practice ofthe present invention include, though are not limited to, HuMEC BasalSerum free Medium, KNOCKOUT™ CTS™ XenoFREE ESC/iPSC Medium, STEMPRO™-34SFM Medium, STEMPRO™ NSC Medium, ESSENTIAL™-8 Medium, Medium 254,Medium, 106, Medium, 131, Medium, 154, Medium, 171, Medium 171, Medium200, Medium 231, HeptoZYME-SFM, Human Endothelial-SFM, GIBCO® FREESTYLE™293 Expression Medium, Medium 154CF/PRF, Medium 154C, Medium 154 CF,Medium 106, Medium 200PRF, Medium 131, Essential™-6 Medium, STEMPRO™-34Medium, Gibco® Astrocyte Medium, AIM V® Medium CTS™, AMINOMAX™ C-100Basal Medium, AMINOMAX™-II Complete Medium, CD FORTICHO™ Medium, CD CHOAGT Medium, CHO-S-SFM Medium, GIBCO®FREESTYLE™ CHO Expression Medium, CDOPTICHO™ Medium, CD CHO Medium, CD DG44 Medium, SF-900™ Medium, LHCBasal Medium, LHC-8 Medium, 293 SFM Medium, CD 293 Medium, AEM GrowthMedium, PER. C6® Cell Medium, AIM V® Medium, EXPILIFE® Medium,Keratinocyte-SFM Medium, LHC Medium, LHC-8 Medium, LHC-9 Medium, and anyderivatives or modifications thereof. In certain preferred thoughnon-limiting embodiments, a high density culture media may be CDFORTICHO™ Medium, CD CHO AGT Medium, CHO-S-SFM Medium, GIBCO®FREESTYLE™CHO Expression Medium, CD OPTICHO™ Medium, CD CHO Medium, CD DG44Medium, GIBCO® FREESTYLE™ 293 Expression Medium, EXPI293™ ExpressionMedium, or a like medium, or a modified version thereof. The abovelisted exemplary high density culture media may be particularly suitablefor the high density growth, propagation, transfection and maintenanceof CHO cells, a CHO cell variant, 293 cells, a 293 cell variant, CapTcells, a CapT cell variant, or any other cells adapted for use in a highdensity culture system. Optionally, a user may wish to formulate a newculture medium having the properties described herein, or may optinstead to reformulate or modify existing culture media.

In some aspects, a high density growth medium may be selected from thelist Such media include, but are not limited to, CD FORTICHO™ Medium,Expi293™ Expression Media, Dulbecco's Modified Eagle's Medium (DMEM),Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI-1640,Ham's F-10, Ham's F-12, α-Minimal Essential Medium (α-MEM), Glasgow'sMinimal Essential Medium (G-MEM), and Iscove's Modified Dulbecco'sMedium (IMDM). Other media that are commercially available (e.g., fromLife Technologies Corporation, Carlsbad, Calif.) or that are otherwiseknown in the art can be equivalently used in accordance with the presentinvention including, but not limited to, 293 SFM, CD-CHO medium, VP SFM,BGJb medium, Brinster's BMOC-3 medium, cell culture freezing medium,CMRL media, EHAA medium, eRDF medium, Fischer's medium, Gamborg's B-5medium, GLUTAMAX™ supplemented media, Grace's insect cell media, HEPESbuffered media, Richter's modified MEM, IPL-41 insect cell medium,Leibovitz's L-15 media, McCoy's 5A media, MCDB 131 medium, Media 199,Modified Eagle's Medium (MEM), Medium NCTC-109, Schneider's Drosophilamedium, TC-100 insect medium, Waymouth's MB 752/1 media, William's MediaE, protein free hybridoma medium II (PFHM II), AIM V media, KeratinocyteSFM, defined Keratinocyte SFM, STEMPRO® SFM, STEMPRO® completemethylcellulose medium, HepatoZYME-SFM, Neurobasal™ medium, Neurobasal-Amedium, Hibernate™ A medium, Hibernate E medium, Endothelial SFM, HumanEndothelial SFM, Hybridoma SFM, PFHM II, Sf 900 medium, Sf 900 TI SFM,EXPRESS FIVE® medium, CHO-S-SFM, AMINOMAX-II complete medium,AMINOMAX-C100 complete medium, AMINOMAX-C 100 basal medium, PB-MAX™karyotyping medium, KARYOMAX bone marrow karyotyping medium, KNOCKOUTD-MEM and CO₂ independent medium. The above media are obtained frommanufacturers known to those of ordinary skill in the art, such as JRH,Sigma, HyClone, and BioWhittaker. Additional examples of media suitablefor use in the practice of the present invention can be found in U.S.Pat. Nos. 5,135,866 and 5,232,848 as well as in internationalpublications nos. WO 88/02774, WO 98/15614, WO 98/08934 and EuropeanPatent No. 0 282 942, the entireties of which are specificallyincorporated herein by reference. Optionally, a user may wish toformulate a new culture medium having the properties described herein,or may opt instead to reformulate or modify existing culture media.

The invention further provides compositions comprising the culture mediaof the present invention, which optionally can further comprise one ormore mammalian epithelial or fibroblast cells, such as those describedabove, particularly one or more 293 cells, 293 F cells, PER-C6 cells,CHO cells, CapT cells, COS-7L cells and Sp2/0 cells, or any derivativesthereof.

In some aspects of the invention, the high yield transient transfectionsystem of the present invention may include one or more cells or celllines that are or have been adapted to grow under high density conditionwithout substantial loss in their viability, ability to be efficientlytransfected, or their ability to express high levels of recombinantprotein. Preferably, a cell are cell line suitable for use in thepresent invention growth and propagation of cultured mammalian cells atdensities in the range of about 1×10⁶ to about 20×10⁶ cells/ml, morepreferably in the range of about 2×10⁶ to about 6×10⁶. Any cell line maybe used, without limitation, provided the cell line are capable ofgrowing under high density conditions as defined above, whilemaintaining their viability at high density in excess of about 80%, andretaining their ability to transfect at high efficiency and expressrecombinant protein at levels up to about 2 g/L of culture. Theidentification of such a cell line is well within the purview of theskilled artisan, and such a person can identify a suitable cell line foruse in the present invention without departing from the spirit and scopethereof. The cells adapted for high density culture may be a celllineage or a (non-clonal) population of cells derived from the sameparental cell lineage which have been adapted to grow at high density ina high density culture medium while retaining cell viability at or aboveabout 80%. Such cells may be isolated or selected out from the parentalpopulation of cells by maintaining the cells at high densityover >40, >50, >60, >70, or >80 sequential passages and graduallyreplacing the proportion of growth medium with the desired high densityculture medium. Optionally, during the process, different pools of cellsmay be individually propagated and subjected to the selection procedurewhile simultaneously assessing transfection efficiency and or proteinexpression efficiency, so that non-clonal population of cells may beselected that can be sustained and grown at high density, transfectedwith high efficiency, and express high levels of a desired recombinantprotein. While it will be readily apparent to the skilled practitionerthat a variety of cell types and lineages may be subjected to thisselection procedure, it has been determined that cell lineages derivedfrom CHO cells, cell lineages derived from 293 fibroblast cells, andcells derived from CapT cells are particularly amenable to the selectionprocess for being adapted to high density growth conditions. Ideally,cells that are adapted to high density growth culture and amenable foruse in the present invention will also be capable of being transfectedat high efficiency and/or capable of expressing recombinant protein atyield exceeding at least 200 about μg/mL of cell culture up to about 2mg/mL of cell culture, more typically between about 500 μg/ml of cellculture to about 1 mg/mL of cell culture. Ideally, cells adapted forhigh density culture used in accordance with the present invention arecapable of being sustained and transfected at densities in the range ofabout 1×10⁶ to about 20×10⁶ cells/ml, about 2×10⁶ to about 2×10⁶cells/ml, or about 2.5×10⁶ to about 6×10⁶ cells/ml.

By way of non-limiting example, cells or cell lines that may be adaptedfor high density culture according to the embodiments described hereinmay include cell such as cultured eukaryotic cells, more preferably,cultured plant and/or animal cells, more preferably, cultured mammaliancells, fish cells, insect cells, amphibian cells or avian cells. Incertain preferred though non limiting embodiments, cells or cell linesthat may be adapted for high density culture according to theembodiments described herein may include culture mammalian cells,including primary epithelial cells (e.g., keratinocytes, cervicalepithelial cells, bronchial epithelial cells, tracheal epithelial cells,kidney epithelial cells and retinal epithelial cells) and establishedcell lines and their strains (e.g., 293 embryonic kidney cells, BHKcells, HeLa cervical epithelial cells and PER-C6 retinal cells, MDBK(NBL-1) cells, 911 cells, CRFK cells, MDCK cells, CapT cells, CHO cells,BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3cells, Hep-2 cells, KB cells, LS180 cells, LS174T cells, NCI-H-548cells, RPMI 2650 cells, SW-13 cells, T24 cells, WI-28 VA13, 2RA cells,WISH cells, BS-C-I cells, LLC-MK₂ cells, Clone M-3 cells, 1-10 cells,RAG cells, TCMK-1 cells, Y-1 cells, LLC-PK₁ cells, PK(15) cells, GH1cells, GH₃ cells, L2 cells, LLC-RC 256 cells, MH₁C₁ cells, XC cells,MDOK cells, VSW cells, and TH-I, B1 cells, or derivatives thereof),fibroblast cells from any tissue or organ (including but not limited toheart, liver, kidney, colon, intestines, esophagus, stomach, neuraltissue (brain, spinal cord), lung, vascular tissue (artery, vein,capillary), lymphoid tissue (lymph gland, adenoid, tonsil, bone marrow,and blood), spleen, and fibroblast and fibroblast-like cell lines (e.g.,CHO cells, TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells,citrullinemia cells, Dempsey cells, Detroit 551 cells, Detroit 510cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit539 cells, Detroit 548 cells, Detroit 573 cells, HEL 299 cells, IMR-90cells, MRC-5 cells, WI-38 cells, WI-26 cells, MiCl₁ cells, CHO cells,CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, Vero cells,DBS-FrhL-2 cells, BALB/3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB/3T3cells, K-BALB cells, BLO-11 cells, NOR-10 cells, C₃H/IOTI/2 cells,HSDM₁C₃ cells, KLN₂O₅ cells, McCoy cells, Mouse L cells, Strain 2071(Mouse L) cells, L-M strain (Mouse L) cells, L-MTK⁻ (Mouse L) cells,NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss/3T3 cells, Indianmuntjac cells, SIRC cells, C_(II) cells, and Jensen cells, orderivatives thereof). Most preferably, the medium is used to culturemammalian cells selected from the group consisting of 293 cells, 293 Fcells or derivatives thereof, PER-C6 cells or derivatives thereof, CHOcells or derivatives thereof, CapT cells or derivatives thereof, COS-7Lcells or derivatives thereof and Sp2/0 cells or derivatives thereof, orany other suspension cell line or derivative capable of being culturedat high cell density as defined above. More preferably, the medium isused to culture 293 cells or a modified 293 cell line specificallyadapted for optimal growth in the cell culture medium that forms thebasis of the present invention. In some preferred though non-limitingaspects of the present invention, the cells adapted for use inhigh-density culture are suspension cells, or adherent cells that havebeen adapted to grow in suspension.

Cells supported by the medium of the present invention can be derivedfrom any animal, preferably a mammal, and most preferably a mouse or ahuman. The cells cultivated in the present media can be normal cells orabnormal cells (i.e., transformed cells, established cells, or cellsderived from diseased tissue samples).

Cells adapted to high density cultured in accordance with theembodiments described herein may optionally express one or moreexpression-enhancing proteins. As used herein, the term “expressionenhancing protein” refers to any protein expressed by a cell; theexpression of the protein enhances the expression of a recombinantprotein. The expression of an expression-enhancing protein by a cellline or populations of cells may be stable or transient, for thepurposes of the present embodiments. A variety of suchexpression-enhancing proteins are known in the art, and may includeproteins such as, e.g., PKBa, Bcl-x_(L), P21, P18, AKT, and the like. Insome aspects of the invention, the high yield transient transfectionsystem of the present invention may include one or more expressionvectors for transiently expressing a recombinant protein of interest.The expression vector may be provided already containing an expressiblenucleic acid (such as, e.g., a positive control to assess expressionefficiency when compared to an optimized control protein), oralternatively, the expression vector may be provided in a form wherebythe user may easily insert an expressible nucleic acid containing anopen-reading frame of a protein of interest, such that the protein ofinterest can be expressed recombinantly and at high efficiency in thecells.

For recombinant production of a protein of interest, an expressiblenucleic acid encoding the protein is isolated and inserted into areplicable vector for further cloning (amplification of the DNA) or forexpression. DNA encoding the protein may be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of the antibody). Many vectors are available. Thevector components generally include, but are not limited to, one or moreof the following: a signal sequence, an origin of replication, one ormore marker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

a) Signal Sequence Component

A protein of interest may be produced recombinantly not only directly,but also as a fusion polypeptide with a heterologous polypeptide, whichis preferably a signal sequence or other polypeptide having a specificcleavage site at the N-terminus of the mature protein or polypeptide.The heterologous signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. In mammalian cell expression, mammalian signal sequences aswell as viral secretory leaders, for example, the herpes simplex gDsignal, are available.

b) Origin of Replication

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2 plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

c) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take upantibody-encoding nucleic acid, such as DHFR, glutamine synthetase (GS),thymidine kinase, metallothionein-I and -II, preferably primatemetallothionein genes, adenosine deaminase, ornithine decarboxylase,etc.

For example, cells transformed with the DHFR gene are identified byculturing the transformants in a culture medium containing methotrexate(Mtx), a competitive antagonist of DHFR. Under these conditions, theDHFR gene is amplified along with any other co-transformed nucleic acid.A Chinese hamster ovary (CHO) cell line deficient in endogenous DHFRactivity (e.g., ATCC CRL-9096) may be used.

Alternatively, cells transformed with the GS gene are identified byculturing the transformants in a culture medium containing L-methioninesulfoximine (Msx), an inhibitor of GS. Under these conditions, the GSgene is amplified along with any other co-transformed nucleic acid. TheGS selection/amplification system may be used in combination with theDHFR selection/amplification system described above.

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody of interest, wild-type DHFR gene, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135(1990). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed. Fleer et al., Bio/Technology, 9:968-975(1991).

d) Promoter Component

Expression and cloning vectors generally contain a promoter that isrecognized by the host organism and is operably linked to nucleic acidencoding a protein of interest. A variety of promoter sequences areknown for eukaryotes. Virtually all eukaryotic genes have an AT-richregion located approximately 25 to 30 bases upstream from the site wheretranscription is initiated. Another sequence found 70 to 80 basesupstream from the start of transcription of many genes is a CNCAATregion where N may be any nucleotide. At the 3′ end of most eukaryoticgenes is an AATAAA sequence that may be the signal for addition of thepoly A tail to the 3′ end of the coding sequence. All of these sequencesare suitably inserted into eukaryotic expression vectors.

Protein transcription from vectors in mammalian host cells can becontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus, Simian Virus 40(SV40), or from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

e) Enhancer Element Component

Transcription of a DNA encoding a protein of interest in accordance withthe present invention by higher eukaryotes is often increased orenhanced by inserting an enhancer sequence into the vector. Manyenhancer sequences are now known from mammalian genes (globin, elastase,albumin, α-fetoprotein, and insulin). Often, though not exclusively, onewill use an enhancer from a eukaryotic cell virus. Examples include theSV40 enhancer on the late side of the replication origin (bp 100-270),the cytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. See alsoYaniv, Nature 297:17-18 (1982) on enhancing elements for activation ofeukaryotic promoters. The enhancer may be spliced into the vector at aposition 5′ or 3′ to the antibody-encoding sequence, but is preferablylocated at a site 5′ from the promoter. Additional enhancers are knownin art, and may include, for example, enhancers obtained or derived frommammalian or viral genes. One particularly preferred enhancerscontemplated for use herein is the woodchuck hepatitispost-transcriptional regulatory element (WPRE).

f) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding antibody. One useful transcriptiontermination component is the bovine growth hormone polyadenylationregion. See WO94/11026 and the expression vector disclosed therein.

In some aspects, an expression vector well-suited for the practice ofthe present invention may be any of the well-known vectors used in theart, such as, e.g., pCDNA 3.3, or a modified version thereof.Non-limiting examples of the types of modification to a vector that maybe suitable in the practice of the present invention include, though arenot limited to, modification such as the addition of modification of oneor more enhancers, one or more promoters, one or more ribosomal bindingsites, one or more origins of replication, or the like. In certainpreferred though non-limiting embodiments, and expression vector used inthe practice of the present invention may include one or more enhancerelements selected to improve expression of the protein of interest inthe present transient expression system. The selected enhancer elementmay be positioned 5′ or 3′ to the expressible nucleic acid sequence usedto express the protein of interest. A particularly preferred thoughnon-limiting enhancer element is the woodchuck hepatitispost-transcriptional regulatory element (WPRE).

In one preferred though non-limiting embodiment, an expression vectorused in accordance with the presently described invention may be a pcDNAvector, or particularly, a pcDNA 3.3 vector, more particularly a variantof a pcDNA 3.3 vector. The vector may optionally include an enhancedpromoter, such as, e.g., and enhanced CMV promoter. Optionally, thevector may include an Adeno T+M region, optionally an SV40ori site,optionally an SV40 splice donor/acceptor site, or optionally a woodchuckhepatitis post-transcriptional regulatory element (WPRE).

In some aspects of the invention, the high yield transient transfectionsystem of the present invention may include one or more expressionenhancers. An expression enhancer can be an aqueous solution containingone or more compounds that increase expression of a recombinant proteinin a transient expression system. A variety of expression enhancers areknown in the art, and any one or more may be used in the practice of thepresent invention without limitation.

Generally, the one or more transfection enhancers are contacted with apopulation of protein-expressing cells during or after said cells havebeen transfected with an expressible nucleic acid or expression vector.When two or more expression enhancer are used, each expression enhancermay be contacted with the cells at substantially the same time, oralternatively the expression enhancers may be contacted with theprotein-expressing cells sequentially, optionally after a period of timehas passed between contacting the cells with a first expression enhancerand contacting the cells with a second expression enhancer.

While it will be readily appreciated by the skilled artisan that anynumber of expression enhancers may be used in the practice of thepresent invention, without limitation, and the identification of whatconstitutes a suitable expression enhancer for use in the presentembodiments is well within the purview of such a person, a variety ofexemplary though non-limiting expression enhancers will be describedbelow, though it is to be understood that the recitation thereof doesnot limit the scope of suitable expressions that may be contemplated foruse in the practice of the present invention.

In some aspects, one or more expression enhancers may include liquid(preferably aqueous) additives used to supplement a culture mediumformulation in accordance with the presently described embodiments, saidadditives being selected to improve the yield of expressed proteinproduced in a transient protein expression system in accordance with thepresently described embodiments. One or more expression enhancers mayinclude one or more of several compounds that impact cell cycleprogression, inhibit apoptosis, slow cell growth and/or promote proteinproduction. In the context of the present invention, the term“expression enhancers” generally refers to any one or more compoundsadded to a transient transfection system, the presence of which enhancesor promotes expression of a target protein by a factor of at least 2fold up to about 10-fold above the expression level seen in the absenceof such expression enhancer(s). Exemplary expression enhancers suitablefor use with the presently described embodiments include, though are notlimited to, additives such as valproic acid (VPA, acid and sodium salt),sodium propionate, lithium acetate, dimethyl sulfoxide (DMSO), sugarsincluding galactose, amino acid mixtures, or butyric acid, or anycombinations of the aforementioned. The optimal concentration of eachspecific expression enhancer may vary according to individualcharacteristics of the expression system and the requirements of theuser, and the determination of what constitutes an optimal concentrationof any one or more expression enhancer in a given experimental scenariois well within purview of a practitioner having ordinary skill level inthe art.

In one exemplary embodiment, an expression enhancer can be a formulationcontaining valproic acid. The optimal final concentration ranges ofvalproic acid (VPA) used in the practice of the present invention mayvary, but will preferably be in the range of about 0.20 mM to about 25mM, or any sub-ranges or concentration values encompassed by this range.More preferably, the final concentration of VPA may be in the range ofabout 0.25 mM to about 24 mM, about 0.26 mM to about 23 mM, 0.27 mM toabout 23 mM, 0.28 mM to about 23 mM, 0.29 mM to about 22 mM, about 0.30mM to about 21 mM, about 0.31 mM to about 20 mM, about 0.32 mM to about19 mM, about 0.33 mM to about 17 mM, about 0.34 mM to about 18 mM, about0.35 mM to about 17 mM, about 0.36 mM to about 16 mM, about 0.37 mM toabout 15 mM, about 0.40 mM to about 14 mM, about 0.41 mM to about 13 mM,about 0.42 mM to about 12 mM, about 0.43 mM to about 11 mM, about 0.44mM to about 10 mM, about 0.45 mM to about 9 mM, about 0.46 mM to about 8mM, about 0.47 mM to about 7 mM, about 0.48 mM to about 6 mM, about 0.49mM to about 5 mM, about 0.50 mM to about 4 mM, about 0.50 mM to about 4mM, about 0.55 mM to about 3 mM, 0.6 mM to about 2 mM or 0.75 to about1.5 mM. In some preferred though non-limiting embodiments, the finalconcentration of VPA used in the practice of the present invention maybe between about 0.15 mM to about 1.5 mM, about 0.16 mM to about 1.5 mM,about 0.17 mM to about 1.5 mM, about 0.18 mM to about 1.5 mM, about 0.19mM to about 1.5 mM, about 0.20 mM to about 1.5 mM, about 0.25 mM toabout 1.5 mM, about 0.30 mM to about 1.5 mM, about 0.40 mM to about 1.5mM, about 0.50 mM to about 1.5 mM, about 0.60 mM to about 1.5 mM, about0.70 mM to about 1.5 mM, about 0.80 mM to about 1.5 mM, about 0.90 mM toabout 1.5 mM or about 0.10 mM to about 1.5 mM. In some preferred thoughnon-limiting embodiments, the final concentration of VPA used in thepractice of the present invention may be between about 0.20 to about 1.5mM, about 0.21 to about 1.4 mM, about 0.22 to about 1.4 mM, about 0.23to about 1.4 mM, about 0.24 to about 1.4 mM, about 0.25 to about 1.3 mM,about 0.25 to about 1.2 mM, about 0.25 to about 1.1 mM, or about 0.25 toabout 1.0 mM.

In another exemplary embodiment, an expression enhancer can be aformulation containing sodium propionate (NaPP). Optionally, NaPP may beprovided alone or in combination with valproic acid as above. Theoptimal final concentration ranges of NaPP used in the practice of thepresent invention may vary, but will preferably be in the range of aboutIn further embodiments, the optimal final concentration of NaPP used inthe practice of the present invention may be in the range of about 0.2mM to about 100 mM, or any sub-range or individual concentrationencompassed within this range. In certain preferred though non-limitingembodiments, the optimal final concentration of NAPP may be in the rangeof about 0.5 to about 80 mM, about 0.4 mM to about 70 mM, about 0.5 mMto about 60 mM, about 0.6 mM to about 50 mM, about 0.7 mM to about 40mM, about 0.8 mM to about 30 mM, about 0.9 mM to about 20 mM, about 1 mMto about 15 mM, about 2 mM to about 10 mM, about 3 mM to about 9 mM,about 4 mM to about 8 mM, or about 5 mM to about 7 mM. In certainpreferred though non-limiting embodiments, the optimal finalconcentration of NAPP may be in the range of about 1 mM to about 10 mM,about 1 mM to about 2 mM, about 2 mM to about 3 mM, about 3 mM to about4 mM, about 4 mM to about 5 mM, about 5 mM to about 6 mM, about 6 mM toabout 7 mM, about 7 mM to about 8 mM, about 8 mM to about 9 mM, or about9 mM to about 10 mM. In certain preferred though non-limitingembodiments, the optimal final concentration of NAPP may be about 1 mM,about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM,about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, about9.5 mM, or about 10 mM.

In yet another exemplary embodiment, an expression enhancer can be aformulation containing lithium acetate (LiAc). Optionally, LiAc may beprovided alone or in combination with NaPP or valproic acid as above. Infurther embodiments, the optimal final concentration of lithium acetate(LiAc) used in the practice of the present invention may be in the rangeof about 0.25 to about 25 mM, about 0.26 mM to about 20 mM, about 0.27mM to about 15 mM, about 0.28 mM to about 10 mM, about 0.29 mM to about5 mM, about 0.3 mM to about 4.5 mM, about 0.31 mM to about 4 mM, about0.35 mM to about 3 mM, about 0.5 mM to about 2.5 mM, about 1 mM to about3 mM, about 1.5 mM to about 2.5 mM, or about 2 mM to about 3 mM.

In yet another exemplary embodiment still, an expression enhancer can bea formulation containing butyric acid. The optimal final concentrationof butyric acid used in the practice of the present invention may be inthe range of about 0.25 to about 25 mM, about 0.26 mM to about 20 mM,about 0.27 mM to about 15 mM, about 0.28 mM to about 10 mM, about 0.29mM to about 5 mM, about 0.3 mM to about 4.5 mM, about 0.31 mM to about 4mM, about 0.35 mM to about 3 mM, about 0.5 mM to about 2.5 mM, about 1mM to about 3 mM, about 1.5 mM to about 2.5 mM, or about 2 mM to about 3mM.

An expression enhancer used in accordance with the present invention maybe added to the culture medium immediately prior to or duringtransfection, or after transfection but prior to harvesting the cellsand the expressed protein. In some specific though non-limitingembodiments described below, “Enhancer 1” generally refers to 0.25 mM-1mM valproic acid, and “Enhancer 2” generally refers to 5 mM-7 mM sodiumpropionate. However, if indicated otherwise, the terms Enhancer 1 andEnhancer 2 may encompass different enhancer compounds. Expressionenhancers may be added to a culture medium sequentially, or as acocktail.

In some aspects of the invention, the high yield transient transfectionsystem of the present invention may include one or more reagents for theintroduction of macromolecules into the cultured cells (said reagentsbeing commonly referred to as “transfection reagents”). A transfectionreagent used in accordance with the presently described embodiments canbe any compound or other chemical modality for introducing a biologicalmolecule, particularly a nucleic acid molecule, into a cell whereby thenucleic acid may exert a biological function, or in the case of anexpressible nucleic acid, where a gene or protein encoded by saidexpressible nucleic acid can be expressed. A variety of suitabletransfection reagents are known in the art, and any one or more may beused in the practice of the present invention without limitation.

A transfection reagent for use with the present embodiments is anyformulation or composition known to those of skill in the art whichfacilitates the entry of a macromolecule into a cell. For example, seeU.S. Pat. No. 5,279,833. In some embodiments, the reagent can be a“transfection reagent” and can be any compound and/or composition thatincreases the uptake of one or more nucleic acids into one or moretarget cells. A variety of transfection reagents are known to thoseskilled in the art. Suitable transfection reagents can include, but arenot limited to, one or more compounds and/or compositions comprisingcationic polymers such as polyethyleneimine (PEI), polymers ofpositively charged amino acids such as polylysine and polyarginine,positively charged dendrimers and fractured dendrimers, cationicβ-cyclodextrin containing polymers (CD-polymers), DEAE-dextran and thelike. In some embodiments, a reagent for the introduction ofmacromolecules into cells can comprise one or more lipids which can becationic lipids and/or neutral lipids. Preferred lipids include, but arenot limited to, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylamoniumchloride (DOTMA), dioleoylphosphatidylcholine (DOPE),1,2-Bis(oleoyloxy)-3-(4′-trimethylammonio) propane (DOTAP),1,2-dioleoyl-3-(4′-trimethylammonio) butanoyl-sn-glycerol (DOTB),1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC), cholesteryl(4′-trimethylammonio)butanoate (ChoTB), cetyltrimethylammonium bromide(CTAB), 1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide (DORI),1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE),1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide(DMRIE),O,O′-didodecyl-N-[p(2-trimethylammonioethyloxy)benzoyl]-N,N,N-trimethylammoniumchloride, spermine conjugated to one or more lipids (for example,5-carboxyspermylglycine dioctadecylamide (DOGS),N,N^(I),N^(II),N^(III)-tetramethyl-N,N^(I),N^(II),N^(III)-tet-rapalmitylspermine(TM-TPS) and dipalmitoylphasphatidylethanolamine 5-carboxyspermylaminde(DPPES)), lipopolylysine (polylysine conjugated to DOPE), TRIS(Tris(hydroxymethyl)aminomethane, tromethamine) conjugated fatty acids(TFAs) and/or peptides such as trilysyl-alanyl-TRIS mono-, di-, andtri-palmitate, (3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol(DC-Chol), N-(α-trimethylammonioacetyl)-didodecyl-D-glutamate chloride(TMAG), dimethyl dioctadecylammonium bromide (DDAB),2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanamin-iniumtrifluoroacetate(DOSPA) and combinations thereof.

Those skilled in the art will appreciate that certain combinations ofthe above mentioned lipids have been shown to be particularly suited forthe introduction of nucleic acids into cells for example a 3:1 (w/w)combination of DOSPA and DOPE is available from Life TechnologiesCorporation, Carlsbad, Calif. under the trade name LIPOFECTAMINE™, a 1:1(w/w) combination of DOTMA and DOPE is available from Life TechnologiesCorporation, Carlsbad, Calif. under the trade name LIPOFECTIN®, a 1:1(M/M) combination of DMRIE and cholesterol is available from LifeTechnologies Corporation, Carlsbad, Calif. under the trade name DMRIE-Creagent, a 1:1.5 (M/M) combination of TM-TPS and DOPE is available fromLife Technologies Corporation, Carlsbad, Calif. under the trade nameCellFECTIN® and a 1:2.5 (w/w) combination of DDAB and DOPE is availablefrom Life Technologies Corporation, Carlsbad, Calif. under the tradename LipfectACE®. In addition to the above-mentioned lipid combinations,other formulations comprising lipids in admixture with other compounds,in particular, in admixture with peptides and proteins comprisingnuclear localization sequences, are known to those skilled in the art.For example, see international application no. PCT/US99/26825, publishedas WO 00/27795, both of which are incorporated by reference herein.

Lipid aggregates such as liposomes have been found to be useful asagents for the delivery of macromolecules into cells. In particular,lipid aggregates comprising one or more cationic lipids have beendemonstrated to be extremely efficient at the delivery of anionicmacromolecules (for example, nucleic acids) into cells. One commonlyused cationic lipid isN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).Liposomes comprising DOTMA alone or as a 1:1 mixture withdioleoylphosphatidylethanolamine (DOPE) have been used to introducenucleic acids into cells. A 1:1 mixture of DOTMA:DOPE is commerciallyavailable from Life Technologies Corporation, Carlsbad, Calif. under thetrade name of LIPOFECTIN™. Another cationic lipid that has been used tointroduce nucleic acids into cells is1,2-bis(oleoyl-oxy)-3-3-(trimethylammonia) propane (DOTAP). DOTAPdiffers from DOTMA in that the oleoyl moieties are linked to thepropylamine backbone via ether bonds in DOTAP whereas they are linkedvia ester bonds in DOTMA. DOTAP is believed to be more readily degradedby the target cells. A structurally related group of compounds whereinone of the methyl groups of the trimethylammonium moiety is replacedwith a hydroxyethyl group are similar in structure to the Rosenthalinhibitor (RI) of phospholipase A (see Rosenthal, et al., (1960) J.Biol. Chem. 233:2202-2206.). The RI has stearoyl esters linked to thepropylamine core. The dioleoyl analogs of RI are commonly abbreviatedDOR1-ether and DOR1-ester, depending upon the linkage of the lipidmoiety to the propylamine core. The hydroxyl group of the hydroxyethylmoiety can be further derivatized, for example, by esterification tocarboxyspermine.

Another class of compounds which has been used for the introduction ofmacromolecules into cells comprise a carboxyspermine moiety attached toa lipid (see, Behr, et al., (1989) Proceedings of the National Academyof Sciences, USA 86:6982-6986 and EPO 0 394 111). Examples of compoundsof this type include dipalmitoylphosphatidylethanolamine5-carboxyspermylamide (DPPES) and 5-carboxyspermylglycinedioctadecylamide (DOGS). DOGS is commercially available from Promega,Madison, Wis. under the trade name of TRANSFECTAM™.

A cationic derivative of cholesterol(3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol, DC-Chol) hasbeen synthesized and formulated into liposomes with DOPE (see Gao, etal., (1991) BBRC 179(1):280-285.) and used to introduce DNA into cells.The liposomes thus formulated were reported to efficiently introduce DNAinto the cells with a low level of cellular toxicity. Lipopolylysine,formed by conjugating polylysine to DOPE (see Zhou, et al., (1991) BBA1065:8-14), has been reported to be effective at introducing nucleicacids into cells in the presence of serum.

Other types of cationic lipids that have been used to introduce nucleicacids into cells include highly packed polycationic ammonium, sulfoniumand phosphonium lipids such as those described in U.S. Pat. Nos.5,674,908 and 5,834,439, and international application no.PCT/US99/26825, published as WO 00/27795. One particularly preferredthough non-limiting transfection reagent for delivery of macromoleculesin accordance with the present invention is LIPOFECTAMINE 2000™ which isavailable from Life technologies. See U.S. international application no.PCT/US99/26825, published as WO 00/27795. Another preferred thoughnon-limiting transfection reagent suitable for delivery ofmacromolecules to a cell is EXPIFECTAMINE™. Other suitable transfectionreagents include LIOFECTAMINE™ RNAiMAX, LIPOFECTAMINE™ LTX,OLIGOFECTAMINE™, Cellfectin™, INVIVOFECTAMINE™, INVIVOFECTAMINE™ 2.0,and any of the lipid reagents or formulations disclosed in U.S. PatentAppl. Pub. No. 2012/0136073, by Yang et al. (incorporated herein byreference thereto). A variety of other transfection reagents are knownto the skilled artisan and may be evaluated for the suitability thereofto the transient transfection systems and methods described herein.

The present invention is directed, in part, to a high-yield transienttransfection system that supports (a) the introduction of at least onemacromolecule, preferably an expressible nucleic acid molecule, intoeukaryotic cells in culture, (b) the cultivation of cells into which atleast one macromolecule is introduced, and optionally (c) the productionof recombinant protein product or expression of the nucleic acid incells into which at least one macromolecule is introduced, whereinmedium containing the macromolecule does not need to be removed from theculture and replaced with fresh medium after introduction of at leastone macromolecule into cells and prior to cultivation and production ofprotein product or expression of nucleic acid. The transienttransfection system of the present invention, an the use thereof inaccordance with the methods described herein, results in the rapid andreproducible expression of high levels of a protein of interest in acell culture system. Typically, the present transient transfectionsystems and methods are capable of producing recombinant expressedprotein at levels in the range of about 200 μg protein/L of culture toabout 2 g protein/L of culture, depending on the individual expressioncharacteristics of the desired recombinant protein and cell type used.Using the transient transfection system and methods provided for herein,a user may obtain levels of expressed protein that are about 2-fold toup to about 20-fold in excess of what is currently obtainable usingstandard commercially available transient transfection systems. Usingthe transient transfection system and methods provided for herein, auser may obtain levels of expressed protein that is about 2.5-fold,about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, bout 7-fold, about7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, orup to about 10-fold or greater than that seen with contemporarytransient expression systems. For example, using the present transienttransfection system to produce a recombinant protein, a user may obtaina protein yield between about 2-fold up to about 10-fold higher than theprotein yield obtained using a commercially available transienttransfection system optimized for production of recombinant protein insuspension cells, such as, e.g., FREESTYLE™ Expression System. Methods

The present invention further relates to methods for expressing highlevels of a protein of interest. Methods of the invention may includecultivating mammalian cells (particularly those described above and mostparticularly 293 cells, 293 F cells, PER-C6 cells, CHO cells, CapTcells, COS-7L cells and Sp2/0 cells, or any derivatives thereof) insuspension comprising (a) obtaining a mammalian cell to be cultivated insuspension; and (b) contacting the cell with the culture media of theinvention under conditions sufficient to support the cultivation of thecell in suspension, transfecting the cultured cells with an expressiblenucleic acid encoding a protein of interest, contacting the transfectedcells with one or more expression enhancers, culturing the transfectedcells under conditions permissive to the expression of the protein ofinterest for a defined period of time, and harvesting the cells.

The present invention further relates to methods of producing apolypeptide, and to polypeptides produced by these methods, the methodscomprising (a) obtaining a cell, preferably a mammalian cell describedabove and most preferably a 293 cells, 293 F cells, PER-C6 cells, CHOcells, CapT cells, COS-7L cells and Sp2/0 cells, or any derivativesthereof; (b) contacting the cell with a solution comprising a nucleicacid encoding the polypeptide under conditions causing the introductionof the nucleic acid into the cell; and (c) cultivating the cell in theculture medium of the invention under conditions favoring the expressionof the desired polypeptide by the cell.

In one aspect, a method for expressing a recombinant protein inaccording with the present invention may include obtaining a culture ofcells in a high density culture medium. The cells are preferably asuspension culture of 293 cells, 293 F cells, PER-C6 cells, CHO cells,CapT cells, COS-7L cells or Sp2/0 cells, or any derivatives thereof,which cells have been adapted for growth in high density medium. Whileit will be readily appreciated by the skilled artisan that any volume ofcell culture may be used in the practice of the present invention, theculture will typically be from about 200 μl to 100 liters, morepreferably, the cell culture volume is from about 2 ml to about 50liters, most preferably from about 5 ml to about 5 liters. In someaspects, the cell culture volume can be from about 100 ml to about 50liters. More preferably, the cell culture volume is from about 500 ml toabout 50 liters. More preferably, the cell culture volume is from about500 ml to about 25 liters. More preferably, the cell culture volume isfrom about 500 ml to about 10 liters. More preferably, the cell culturevolume is from about 500 ml to about 5 liters. More preferably, the cellculture volume is from about 500 ml to about 1 liter. In someembodiments, the cell culture volume can be up to about 100 liters, upto about 95 liters, up to about 90 liters, up to about 85 liters, up toabout 80 liters, up to about 75 liters, up to about 70 liters, up toabout 65 liters, up to about 60 liters, up to about 55 liters, up toabout 50 liters, up to about 45 liters, up to about 40 liters, up toabout 35 liters, up to about 30 liters, up to about 35 liters, up toabout 20 liters, up to about 15 liters, up to about 10 liters, up toabout 9 liters, up to about 8 liters, up to about 7 liters, up to about6 liters, up to about 5 liters, up to about 4 liters, up to about 2liters or up to about 1 liter.

In one aspect, the cell culture may be maintained at a cell density ofbetween about 1.5×10⁶ cells/ml to about 20×10⁶ cells/ml, or anyconcentration, concentration range or sub-range encompassed therein.

To express a protein in cells in accordance with the presently describedinvention, the cells will typically be diluted into a fresh volume ofmedium. The optimal dilution can vary, though for illustrative purposes,the density of cells diluted into a fresh volume of medium can bebetween 0.5×10⁶ cells/ml to about 10×10⁶ cells/ml, more preferably 1×10⁶cells/ml to about 5×10⁶ cells/ml, more preferably, 1.5×10⁶ cells/ml toabout 3×10⁶ cells/ml.

In one aspect, following dilution of the cells into a fresh volume ofculture medium, the cells can be cultured in said volume for a period oftime, prior to being transfected with an expressible nucleic acid.Optionally, the cells can be cultured for up to 2 days, more preferablyup to about a day and a half, most preferably, up to about a day.Optionally, the cells can be cultured in the fresh volume of mediumuntil the density of the cells cultured therein has increased by up toabout 100%, more preferably up to about 95%, up to about 90%, up toabout 85%, up to about 80%, up to about 75%, up to about 70%, up toabout 65%, up to about 60% up to about 55%, up to about 50%, up to about45%, up to about 40%, up to about 35%, up to about 30%, up to about 25%,up to about 20% or up to about 15%.

In one aspect, cells may be transfected with an expressible nucleic acidor an expression vector after the cells have been cultured in the highdensity growth media for a period of time as described above. Theprecise sequence of steps a user undertakes to accomplish theintroduction of the expression vector into the cells may vary, dependingon the specific transfection reagent selected, the cell line, the mediaand various other experimental parameters, as will be readily recognizedby a practitioner having ordinary skill level in the art. By way ofexample only, in the case where a lipid-based transfection system isselected (in particular, a transfection system having at least onecationic lipid), the transfection reagent will first be contacted withthe nucleic acid in an aqueous solution to form lipid-DNA complexes in aprocess known informally as “complexation” or a “complexation reaction”as defined above and incorporated herein. Such a reaction will typicallybe accomplished in a separate reaction vessel from that in which thecells are being cultured.

In an aspect, following the formation of lipid-DNA complexes in thecomplexation step described above, the transfection complexes can becontacted with the cultured cells. After contacting the cells with thetransfection complexes, the cells can be cultured in the presence of thetransfection complexes for a first period of time. The duration of thefirst period of time will vary according to the nature of the cells, thetransfection reagent used, and a variety of other factors know to thoseskilled in the art. The phrase “first period of time”, when used in thecontext of a method for transiently transfecting cells in accordancewith the methods of the invention described herein generally refers tothe time interval between transfecting a population of cells with anexpressible nucleic acid and the additional of one or more expressionenhancers to the transfected cells. Typically, a first period of timewill be in the range of about 2 hrs to about 4 days, or any ranges orsub-ranges encompassed therein. In certain preferred though non-limitingembodiments, a first period of time may be in the range of about 3 toabout 90 hrs, about 4 to about 85 hr, about 5 to about 80 hrs, about 6to about 75 hrs, about 7 to about 70 hrs, about 8 to about 65 hrs, about9 to about 60 hrs, about 10 to about 55 hrs, about 11 to about 50 hrs,about 12 to about 45 hrs, about 13 to about 40 hrs, about 14 to about 35hrs, about 15 to 30 hrs, about 16 to about 24 hrs, about 17 to about 24hrs, about 18 to about 24 hrs, about 19 to about 24 hrs, about 20 toabout 24 hrs, about 21 to about 24 hrs, about 22 to about 24 hrs orabout 23 to about 24 hrs. In other preferred to non-limitingembodiments, a first period of time may be up to about 15 hrs, up toabout 16 hrs, up to about 17 hrs, up to about 18 hrs, up to about 19hrs, up to about 20 hrs, up to about 21 hrs, up to about 22 hrs, up toabout 23 hrs, up to about 24 hrs, up to about 25 hrs, up to about 26hrs, up to about 27 hrs, up to about 28 hrs, up to about 29 hrs or up toabout 30 hrs.

In one highly preferred though non-limiting embodiment, the culturemedium is not replaced, supplemented or replenished following theintroduction of the transfection complexes to the cells, and for theduration of the first period of time.

In one aspect of the present invention, the transfected cells in culturemay be contacted with one or more expression enhancers following thefirst period of time. An expression enhancer can be an aqueous solutioncontaining one or more compounds that increase expression of arecombinant protein in a transient expression system. A variety ofexpression enhancers are known in the art, and any one or more may beused in the practice of the present invention without limitation.

Generally, the one or more transfection enhancers are contacted with apopulation of protein-expressing cells during or after said cells havebeen transfected with an expressible nucleic acid or expression vector.When two or more expression enhancer are used, each expression enhancermay be contacted with the cells at substantially the same time, oralternatively the expression enhancers may be contacted with theprotein-expressing cells sequentially, optionally after a period of timehas passed between contacting the cells with a first expression enhancerand contacting the cells with a second expression enhancer.

While it will be readily appreciated by the skilled artisan that anynumber of expression enhancers may be used in the practice of thepresent invention, without limitation, and the identification of whatconstitutes a suitable expression enhancer for use in the presentembodiments is well within the purview of such a person, a variety ofexemplary though non-limiting expression enhancers will be describedbelow, though it is to be understood that the recitation thereof doesnot limit the scope of suitable expressions that may be contemplated foruse in the practice of the present invention.

In some aspects, one or more expression enhancers may include liquid(preferably aqueous) additives used to supplement a culture mediumformulation in accordance with the presently described embodiments, saidadditives being selected to improve the yield of expressed proteinproduced in a transient protein expression system in accordance with thepresently described embodiments. One or more expression enhancers mayinclude one or more of several compounds that impact cell cycleprogression, inhibit apoptosis, slow cell growth and/or promote proteinproduction. In the context of the present invention, the term“expression enhancers” generally refers to any one or more compoundsadded to a transient transfection system, the presence of which enhancesor promotes expression of a target protein by a factor of at least 2fold up to about 10-fold above the expression level seen in the absenceof such expression enhancer(s). Exemplary expression enhancers suitablefor use with the presently described embodiments include, though are notlimited to, additives such as valproic acid (VPA, acid and sodium salt),sodium propionate, lithium acetate, dimethyl sulfoxide (DMSO), sugarsincluding galactose, amino acid mixtures, or butyric acid, or anycombinations of the aforementioned. The optimal concentration of eachspecific expression enhancer may vary according to individualcharacteristics of the expression system and the requirements of theuser, and the determination of what constitutes an optimal concentrationof any one or more expression enhancer in a given experimental scenariois well within purview of a practitioner having ordinary skill level inthe art.

In one exemplary embodiment, an expression enhancer can be a formulationcontaining valproic acid. The optimal final concentration ranges ofvalproic acid (VPA) used in the practice of the present invention mayvary, but will preferably be in the range of about 0.20 mM to about 25mM, or any sub-ranges or concentration values encompassed by this range.More preferably, the final concentration of VPA may be in the range ofabout 0.25 mM to about 24 mM, about 0.26 mM to about 23 mM, 0.27 mM toabout 23 mM, 0.28 mM to about 23 mM, 0.29 mM to about 22 mM, about 0.30mM to about 21 mM, about 0.31 mM to about 20 mM, about 0.32 mM to about19 mM, about 0.33 mM to about 17 mM, about 0.34 mM to about 18 mM, about0.35 mM to about 17 mM, about 0.36 mM to about 16 mM, about 0.37 mM toabout 15 mM, about 0.40 mM to about 14 mM, about 0.41 mM to about 13 mM,about 0.42 mM to about 12 mM, about 0.43 mM to about 11 mM, about 0.44mM to about 10 mM, about 0.45 mM to about 9 mM, about 0.46 mM to about 8mM, about 0.47 mM to about 7 mM, about 0.48 mM to about 6 mM, about 0.49mM to about 5 mM, about 0.50 mM to about 4 mM, about 0.50 mM to about 4mM, about 0.55 mM to about 3 mM, 0.6 mM to about 2 mM or 0.75 to about1.5 mM. In some preferred though non-limiting embodiments, the finalconcentration of VPA used in the practice of the present invention maybe between about 0.15 mM to about 1.5 mM, about 0.16 mM to about 1.5 mM,about 0.17 mM to about 1.5 mM, about 0.18 mM to about 1.5 mM, about 0.19mM to about 1.5 mM, about 0.20 mM to about 1.5 mM, about 0.25 mM toabout 1.5 mM, about 0.30 mM to about 1.5 mM, about 0.40 mM to about 1.5mM, about 0.50 mM to about 1.5 mM, about 0.60 mM to about 1.5 mM, about0.70 mM to about 1.5 mM, about 0.80 mM to about 1.5 mM, about 0.90 mM toabout 1.5 mM or about 0.10 mM to about 1.5 mM. In some preferred thoughnon-limiting embodiments, the final concentration of VPA used in thepractice of the present invention may be between about 0.20 to about 1.5mM, about 0.21 to about 1.4 mM, about 0.22 to about 1.4 mM, about 0.23to about 1.4 mM, about 0.24 to about 1.4 mM, about 0.25 to about 1.3 mM,about 0.25 to about 1.2 mM, about 0.25 to about 1.1 mM, or about 0.25 toabout 1.0 mM.

In another exemplary embodiment, an expression enhancer can be aformulation containing sodium propionate (NaPP). Optionally, NaPP may beprovided alone or in combination with valproic acid as above. Theoptimal final concentration ranges of NaPP used in the practice of thepresent invention may vary, but will preferably be in the range of aboutIn further embodiments, the optimal final concentration of NaPP used inthe practice of the present invention may be in the range of about 0.2mM to about 100 mM, or any sub-range or individual concentrationencompassed within this range. In certain preferred though non-limitingembodiments, the optimal final concentration of NAPP may be in the rangeof about 0.5 to about 80 mM, about 0.4 mM to about 70 mM, about 0.5 mMto about 60 mM, about 0.6 mM to about 50 mM, about 0.7 mM to about 40mM, about 0.8 mM to about 30 mM, about 0.9 mM to about 20 mM, about 1 mMto about 15 mM, about 2 mM to about 10 mM, about 3 mM to about 9 mM,about 4 mM to about 8 mM, or about 5 mM to about 7 mM. In certainpreferred though non-limiting embodiments, the optimal finalconcentration of NAPP may be in the range of about 1 mM to about 10 mM,about 1 mM to about 2 mM, about 2 mM to about 3 mM, about 3 mM to about4 mM, about 4 mM to about 5 mM, about 5 mM to about 6 mM, about 6 mM toabout 7 mM, about 7 mM to about 8 mM, about 8 mM to about 9 mM, or about9 mM to about 10 mM. In certain preferred though non-limitingembodiments, the optimal final concentration of NAPP may be about 1 mM,about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM,about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, about9.5 mM, or about 10 mM.

In yet another exemplary embodiment, an expression enhancer can be aformulation containing lithium acetate (LiAc). Optionally, LiAc may beprovided alone or in combination with NaPP or valproic acid as above. Infurther embodiments, the optimal final concentration of lithium acetate(LiAc) used in the practice of the present invention may be in the rangeof about 0.25 to about 25 mM, about 0.26 mM to about 20 mM, about 0.27mM to about 15 mM, about 0.28 mM to about 10 mM, about 0.29 mM to about5 mM, about 0.3 mM to about 4.5 mM, about 0.31 mM to about 4 mM, about0.35 mM to about 3 mM, about 0.5 mM to about 2.5 mM, about 1 mM to about3 mM, about 1.5 mM to about 2.5 mM, or about 2 mM to about 3 mM.

In yet another exemplary embodiment still, an expression enhancer can bea formulation containing butyric acid. The optimal final concentrationof butyric acid used in the practice of the present invention may be inthe range of about 0.25 to about 25 mM, about 0.26 mM to about 20 mM,about 0.27 mM to about 15 mM, about 0.28 mM to about 10 mM, about 0.29mM to about 5 mM, about 0.3 mM to about 4.5 mM, about 0.31 mM to about 4mM, about 0.35 mM to about 3 mM, about 0.5 mM to about 2.5 mM, about 1mM to about 3 mM, about 1.5 mM to about 2.5 mM, or about 2 mM to about 3mM.

An expression enhancer used in accordance with the present invention maybe added to the culture medium immediately prior to or duringtransfection, or after transfection but prior to harvesting the cellsand the expressed protein. In some specific though non-limitingembodiments described below, “Enhancer 1” generally refers to 0.25 mM-1mM valproic acid, and “Enhancer 2” generally refers to 5 mM-7 mM sodiumpropionate. However, if indicated otherwise, the terms Enhancer 1 andEnhancer 2 may encompass different enhancer compounds. Expressionenhancers may be added to a culture medium sequentially, or as acocktail.

In one aspect, when two or more expression enhancers are used, the twoor more expression enhancers can be contacted with the transfectedcultured cells substantially simultaneously, or alternatively thetransfected cultured cells can first be contacted with a firstexpression enhancer, and after a second period of time, the transfectedcultured cells can be contacted with the second expression enhancer. Inone aspect, the “second period of time”, when used in the context of amethod for transiently transfecting cells in accordance with the methodsof the invention described herein generally refers to the time intervalbetween the addition of one or more expression enhancers and either theaddition of one or more additional enhancers, or the harvesting of thetransfected cells and purification or isolation of the protein expressedtherein. Typically, a second period of time will be in the range ofabout 10 hrs to about 10 days, though other time intervals may be usedif determined to be optimal for the protein being expressed. In somepreferred though non-limiting embodiments, the second period of time maybe in the range of 2 hrs to 5 days, 2.5 hrs to 4 days, about 3 to about90 hrs, about 4 to about 85 hr, about 5 to about 80 hrs, about 6 toabout 75 hrs, about 7 to about 70 hrs, about 8 to about 65 hrs, about 9to about 60 hrs, about 10 to about 55 hrs, about 11 to about 50 hrs,about 12 to about 45 hrs, about 13 to about 40 hrs, about 14 to about 35hrs, about 15 to 30 hrs, about 16 to about 24 hrs, about 17 to about 24hrs, about 18 to about 24 hrs, about 19 to about 24 hrs, about 20 toabout 24 hrs, about 21 to about 24 hrs, about 22 to about 24 hrs orabout 23 to about 24 hrs. In other preferred to non-limitingembodiments, a first period of time may be up to about 15 hrs, up toabout 16 hrs, up to about 17 hrs, up to about 18 hrs, up to about 19hrs, up to about 20 hrs, up to about 21 hrs, up to about 22 hrs, up toabout 23 hrs, up to about 24 hrs, up to about 25 hrs, up to about 26hrs, up to about 27 hrs, up to about 28 hrs, up to about 29 hrs or up toabout 30 hrs.

After an appropriate amount of time has elapsed, the user can harvestthe cells and optionally purify the expressed recombinant protein.

The method of the present invention allows a user to transiently expressa recombinant protein in accordance with the embodiments described abovewithout having to replace, supplement or otherwise replenish the culturemedium during the process. The methods described herein allow the userexpress up to about 2 g/L of cultured cells. In some embodiments, theuser can express up to about 1.9 g, up to about 1.8 g, up to about 1.7g, up to about 1.6 g, up to about 1.5 g, up to about 1.4 g, up to about1.3 g, up to about 1.2 g, up to about 1.1 g, or up to about 1 g ofrecombinant protein for every liter of cultured cells.

The present invention is also directed to compositions, particularly ahigh density cell culture media as defined above, optionally comprisingone or more replacement compounds. The invention is also directed tomethods of use of such compositions, including, for example, methods forthe cultivation of eukaryotic cells, particularly animal cells, invitro. The invention also relates to compositions comprising suchculture media and one or more cells, especially those cells specificallyreferenced herein, and to kits comprising one or more of theabove-described compositions. The invention also relates to expressionvectors comprising one or more expressible nucleic acid sequences incombination with one or more promoters, enhancers, and other elementsrequired for expressing said expressible nucleic acid in a culturedcells, as defined above and incorporated herein. The invention alsorelates to compositions comprising one or more expression enhancercompositions, especially those selected to enhance expression of saidexpressible nucleic acid in a cultured cell by at least a factor or 2-to 2.5 fold. Optionally, the expression enhancers can be a combinationof two or expression enhancers co-formulated or provided separately. Theinvention also relates to transfections reagents, especially thoseoptimized to facilitate the delivery of one or more nucleic acidmolecules to the interior of a cultured cell. The invention also relatesto kits comprising one or more of the above-described compositions,vectors, expression enhancers, transfection reagents, and the like, andto kits comprising one or more of the above-described compositions,especially those cells specifically referenced herein.

In another aspect, the invention relates to a kit for the cultivation ofcells in vitro. The kit comprise one or more containers, wherein a firstcontainer contains the culture medium of the present invention. The kitcan further comprise one or more additional containers, each containercontaining one or more supplements selected from the group consisting ofone or more cytokines, heparin, one or more animal or animal-derivedpeptides, one or more yeast peptides and one or more plant peptides(which are preferably one or more peptides from rice, aloevera, soy,maize, wheat, pea, squash, spinach, carrot, potato, sweet potato,tapioca, avocado, barley coconut and/or green bean, and/or one or moreother plants).

The kit of the present invention can further comprise one or morecontainers comprising a nucleic acid and/or a reagent that facilitatesthe introduction of at least one macromolecule, e.g., a nucleic acidinto cells cultured in the media of the present invention, i.e., atransfection reagent. Preferred transfection reagents include, but arenot limited to, cationic lipids and the like.

A kit according to one aspect of the invention can comprise one or moreof the culture media of the invention, one or more replacementcompounds, which can be one or more metal binding compounds, and/or oneor more transition element complexes, and can optionally comprise one ormore nucleic acids and transfection reagents. Kits according to anotheraspect of the invention can comprise one or more cell culture media (oneof which can be a basal medium) and optionally one or more replacementcompounds. The kit of the present invention can also containinstructions for using the kit to culture cells and/or introducemacromolecules or compounds (e.g., nucleic acid, such as DNA), intocells.

It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein are obvious and can be made withoutdeparting from the scope of the invention or any embodiment thereof.Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention.

EXAMPLES

Objectives:

To develop a cell culture medium and transfection system to maximizeprotein yields (at least 2-fold in excess of that obtained withcommercially available transient expression systems, such as, e.g.,Freestyle™ 293 system). The system should work for multiple proteintypes and in a variety of suspension cells. The system should increasereproducibility and minimize variability and should be scalable(multi-well plates to large scale). Further embodiments of the presentinvention include the development of an improved expression vector, ahigh density cell line adapted for growth under high density cultureconditions in the culture system of the present invention, the use andincorporation of transfection enhancers such, for example, as valproicacid and sodium propionate (among others). It is a further object of thepresent invention to develop a protocol to enable transfection at highcell density, that does not involve media exchange during or aftertransfection, and that is simple and easy to use.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub combination.

Example 1: High Density Culture Medium

A variety of commercially available serum-free, protein-free culturemedia were assessed for their ability to sustain the viability of anadapted 293 F cell line with cell densities up to about 14×10⁶ cells/mland thus be used in the practice of the present invention. A serum-free,protein-free medium was selected wherein the viability of the culturecell line over a time frame exceeding a week remains high and evenapproached densities of nearly 15×10⁶ cells/ml, while also enablingtransfection at surprisingly high cell densities of around 3×10⁶cells/ml (vs. 1×10⁶ cells/ml for present commercially availabletransient transfection systems). The results are depicted in FIG. 1,which shows a graph of the resulting cell densities that are achievableusing the transient transfection system in accordance with someembodiments of the invention. Cells that were previously adapted forhigh density growth were slowly adapted into various tested growth mediaover 3 passages. The media to which the cells were adapted include HighDensity Culture Media in accordance with one embodiment of the invention(closed circles), Test Media 1 (closed triangles), Test Media 2 (opentriangles), and Test Media 3 (open diamonds). Cells were cultured formultiple passages in each of the media before being seeded in 30 mlflasks at 0.2×10⁶ cells/ml. Cell density and viability were monitoredover 8 days without replenishing, replacing or otherwise supplementingthe growth medium over the course of the experiment. One of the selectedgrowth media (High Density Growth Medium; closed circles) was able tosustain a surprisingly high density of cultured suspension cells withoutsubstantially losing viability over the course of the experiment. Thus,it is possible for one skilled in the art to readily assess a variety ofgrowth media for use with a specific cell line are variant of a cellline, where a growth medium can be selected based on the ability tofacilitate the cultivation of high densities of suspension cells over adefined period of time, without having to replace, supplement orreplenish the medium. Such may be accomplished by the skilled artisanwithout undue experimentation.

Example 2: Cell Line Optimization

Although a variety of suspension cells can be used in the practice ofthe present invention, it is preferable to use a cell line that has beenadapted for use with the present embodiments and in the selected highdensity growth medium. Additionally, the cells may be specificallyselected for high density growth, high viability, and increased proteinexpression. To accomplish this, parental 293F fibroblast cells underwentan extensive adaptation process involving gradual media replacement overseveral passages. Additionally, it was noted that the adapted cells'size and expression ability increased with subsequent generations. Atpassage 72, cells were banked and validated through genetic analysis atATCC, and were authenticated as 293F cells with no mycoplasmacontamination. The cells were thawed and their viability was verified.The cells were passaged for 30 passages to verify stability, expressionperformance, and their ability to retain high viability when grown inculture and densities of up to about 20×10⁶ cells/ml. Cell populationsselected for increased cell density, viability, and human IgG expressionas described above exhibited approximately 1.7-fold more hIgG than theoriginal cell line (Line 1). High yield adapted cells (Line 3) also haveincreased growth rate, viability and cell size.

FIG. 2 shows a bar graph outlining cell line expression optimization foruse with a transient transfection system in accordance with someembodiments of the invention. A parental 293F cell line was slit intomultiple subcultures which were subsequently adapted into a High DensityCulture Media. Various subcultures that were able to grown at highdensity were then selected and assessed for their ability to express arecombinant test protein (human IgG). The subculture of cells markedHigh Yield Adapted 293F Cells (right set of bars) expressed between 35%to 45% more recombinant IgG than two different subcultures of cellsderived from the same parental 293F cell line. Thus, it is possible forone skilled in the art to readily obtain a cell line or a derivative ofa cell line that has been specifically selected for use with a growthmedium, and can be selected based on the ability to facilitate thecultivation of high densities of suspension cells over a defined periodof time, without having to replace, supplement or replenish the medium.Such may be accomplished by the skilled artisan without undueexperimentation.

Example 3: Transient Transfection is Aided by Expression Enhancers

A panel of chemical additives was tested in combinatorial experiments toevaluate the relative contribution of each component, or the combinationof one or more components, to protein yield. A variety oftransfection/expression enhancers were identified that significantlyimproved protein production. Components were formulated into 2 stableEnhancer solutions. Transfection Enhancer 1 (valprioc acid, as definedabove) doubles hIgG expression. Enhancer 2 (sodium propionate as definedabove) has no strong effect alone, but in combination with Enhancer 1,provides almost 3 fold more hIgG vs. control (with neither Enhancer 1nor Enhancer 2).

FIG. 3 shows a bar graph outlining the effects of various Enhancer 1 andEnhancer 2 used in a transient transfection system in accordance withsome embodiments. Components were formulated into 2 stable Enhancersolutions. The addition of Expression Enhancer 1 doubles hIgG expression(compare first two bars). The addition of Enhancer 2 by itself showsonly marginal effect on enhancing expression of IgG, but when added incombination with Enhancer 1, provides almost 3 fold more hIgG vs.control (Compare third and fourth

Example 4: Protein Expression Results

The transient expression system of the present invention can producebetween 1 g/L up to about 2 g/L of human IgG and Cripto. The transientexpression system of the present invention system showed between a3.5×-11.8× increase in transient protein expression of the proteinsshown in FIGS. 4A through 4D when compared to the commercially availableFreestyle™ 293 system. FIG. 4 shows a comparison of the expressionlevels of 4 different and unique proteins using a high yield transienttransfection system in accordance with some embodiments and a prior arttransient transfection system (Freestyle™ 293 system). FIG. 4A shows agreater than 5-fold increase in expression of human IgG using thetransient transfection system according to some embodiments of thepresent invention when compared to commercially available FreeStyle™ Maxsystem. FIG. 4B shows a greater than 5.2-fold increase in expression ofCripto using the transient transfection system according to someembodiments of the present invention when compared to commerciallyavailable FreeStyle™ Max system. FIG. 4C shows a almost 4-fold increasein expression of 32-adrenergic receptor using the transient transfectionsystem according to some embodiments of the present invention whencompared to commercially available FreeStyle™ Max system. FIG. 4D showsa greater than 11-fold increase in expression of rabbit IgG using thetransient transfection system according to some embodiments of thepresent invention when compared to commercially available FreeStyle™ Maxsystem.

Example 5: EPO Expression, Scalability and Reproducibility

Next, we sought to examine the scalability and reproducibility of thetransient transfection system using a widely used expressed protein ofclinical importance. Erythropoietin (EPO) was expressed using thetransient transfection system of the present invention (bars on rightside of graph) and Freestyle™ 293 system (bars on left side of graph).The inventive system is scalable from 1 ml (in 24-well plate format) upto 1 L (3 L shake flask format). Very good reproducibility was seen inresults from three separate analysts in three different labs.

CONCLUSIONS

1 g/L expression of two different proteins was achieved using the highyield transient transfection system. High density culture media enabledhigh density transfection. Significant improvements to transient proteinexpression were obtained via cell line selection. Transfection enhancersimprove transfection and expression at high cell densities. The resultsare very scalable and reproducible. The transient transfection system ofthe present invention achieved 3.5×-15-fold increase in proteinexpression compared to Freestyle™ 293 system.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. In caseof conflict, the specification herein, including definitions, willcontrol. Citation or identification of any reference in this applicationshall not be construed as an admission that such reference is availableas prior art to the present invention.

What is claimed is:
 1. A method for producing a recombinant protein incultured 293 cells, said method comprising: obtaining a suspensionculture comprising 293 cells in a high density culture medium, saidsuspension culture having a cell density of between about 2×10⁶ to about2×10⁷ cells/ml; transfecting said 293 cells with an expression vectorcontaining a genetic sequence capable of producing an expressed protein;incubating said transfected 293 cells for a first period of time;contacting said transfected 293 cells with at least one expressionenhancer composition comprising valproic acid or a salt thereof;incubating said transfected 293 cells in the presence of said expressionenhancer composition for a second period of time such that said vectorexpresses said protein; and harvesting said transfected 293 cells aftersaid second period of time; wherein said high density culture medium isnot replaced, replenished, or supplemented with fresh media followingthe transfection step.
 2. The method according to claim 1, wherein saidcultured 293 cells are a suspension culture adapted for growth underhigh density conditions.
 3. The method according to claim 1, whereinsaid 293 cells have been adapted for growth under high densityconditions.
 4. The method according to claim 3, wherein said 293 cellsare 293 F cells.
 5. The method according to claim 1, wherein the volumeof the suspension culture is in the range of about 200 μL to about 5 L.6. The method according to claim 1, wherein said expression enhancercomposition further comprises at least one of sodium propionate, lithiumacetate, dimethyl sulfoxide (DMSO), galactose, amino acid mixtures,butyric acid, or any salts or combinations of the aforementioned.
 7. Themethod according to claim 1, wherein the concentration of valproic acid(VPA) is in the range of about 0.20 mM to about 25 mM.
 8. The methodaccording to claim 1, wherein the concentration of valproic acid is inthe range of about 0.25 mM to about 24 mM.
 9. The method according toclaim 1, wherein the expression composition further comprises sodiumpropionate.
 10. The method according to claim 9, wherein the finalconcentration of sodium propionate in the culture is in the range ofabout 0.2 mM to about 100 mM.
 11. The method according to claim 9,wherein the final concentration of sodium propionate in the culture isin the range of about 0.5 to about 80 mM.
 12. The method according toclaim 1, wherein the expression enhancer composition further compriseslithium acetate (LiAc).
 13. The method according to claim 12, whereinthe final concentration of LiAc in the culture is in the range of about0.25 to about 25 mM.
 14. The method according to claim 12, wherein thefinal concentration of LiAc in the culture is in the range of about 0.26mM to about 20 mM.
 15. The method according to claim 1, wherein theexpression enhancer composition further comprises butyric acid.
 16. Themethod according to claim 15, wherein the final concentration of butyricacid in the culture is in the range of about 0.25 to about 25 mM. 17.The method according to claim 1, wherein the volume of the suspensionculture is in the range of about 25 mL to about 50 L.
 18. The methodaccording to claim 1, wherein the volume of the suspension culture is inthe range of about 100 mL to about 1 L.
 19. The method according toclaim 1, wherein the volume of the suspension culture is in the range ofabout 200 mL to about 500 mL.
 20. The method according to claim 1,wherein the cell density of the transfection step is between about 3×10⁶to about 20×10⁶ cells/ml.
 21. The method according to claim 1, whereinthe cell density of the transfection step is in the range of about 2×10⁶to about 6×10⁶.
 22. The method according to claim 1, wherein saidexpression vector comprises a woodchuck hepatitis virusposttranscriptional regulatory (WPRE) element.
 23. The method accordingto claim 1, wherein said high density culture medium is aserum-free/protein-free chemically defined culture medium capable ofpromoting the growth of transfected cells at cell densities in excess of2.5×10⁶ cells/ml with cell viability remaining in excess of 80%.
 24. Themethod according to claim 1, further comprising purifying said expressedprotein.
 25. The method according to claim 1, wherein said cells expressone or more expression enhancing proteins.
 26. The method according toclaim 25, wherein said expression enhancing proteins are selected fromthe list consisting of AKT, P18, P21, Bcl-X_(L), and PKBa.
 27. Themethod according to claim 25, wherein said cells transiently express oneor more expression enhancing proteins.
 28. The method according to claim25, wherein said cells stably express one or more expression enhancingproteins.
 29. The method according to claim 1, wherein said first periodof time is in the range of about 2 hrs to about 4 days.
 30. The methodaccording to claim 1, wherein said second period of time is in the rangeof about 10 hrs to about 10 days.